BOSTON UNIVERSITY SCHOOL of MEDICINE Dissertation

BOSTON UNIVERSITY

SCHOOL OF MEDICINE

Dissertation

DEAMIDATION AND RELATED PROBLEMS IN STRUCTURAL ANALYSIS OF

PEPTIDES AND PROTEINS

NADEZDA P. SARGAEVA

B.S., St. Petersburg State Polytechnical University, 2002

M.S., St. Petersburg State Polytechnical University, 2004

Submitted in partial fulfillment of the

requirements for the degree of

Doctor of Philosophy

2012

Approved by

First Reader

Peter B. O’Connor, Ph.D.

Associate Professor of Biochemistry

Second Reader

Cheng Lin, Ph.D.

Assistant Professor of Biochemistry

Dedications

I dedicate this thesis to my Family

iii

Acknowledgments

I would like to express my deepest gratitude to my advisor, Prof. Peter B.

O’Connor. We met in St. Petersburg, Russia in 2004 and you believed in my abilities and you gave me an opportunity to be a part of such a great mass spectrometry program. I also want to thank you for your support and your guidance throughout my

Ph.D. studies. Thank you for pushing me to achieve my goals of learning and becoming a better scientist, and for the opportunity you gave me to build up my professional and scientific network. Your help, your advice, and your encouragement in building up my professional career are very much appreciated.

Prof. Cheng Lin, thank you for being my second reader and mentor. Working with

you has always been a pleasure – I highly value your well thought out ideas, your

constant use of the scientific principle, and your willingness to follow up regarding the

data, the future directions, etc. I truly appreciate your attention to details, your regular

availability and willingness to help me hands on. Thank you for mentoring me not just

throughout the program but also regarding the life journey I’m about to embark on.

Peter and Cheng, you are the two most influential people that helped me achieve

my goals and I thank you very much from the bottom of my heart.

I would like to express my gratitude to the other members of my thesis advisory

committee: Prof. Carmela Abraham, Prof. Matthew Nugent, Prof. Mark E. McComb, and

Prof. Joseph Zaia for spending your valuable time serving on my committee and giving

me constructive criticism and advice. Special thanks to Mark (along with Nancy

Leymarie), for spending a lot of time with me working on the last challenging project –

your contribution is very much appreciated.

I am sincerely thankful to Prof. Catherine E. Costello. Your wealth of experience and, more importantly your willingness to share it, have been very important to my development. Thank you for your valuable advice and your contribution to my studies.

The research groups of Prof. Dieter Seebach, Prof. Sam Gellman, Prof. Kaj

Blennow, and Prof. Mikhail Gorshkov are highly appreciated for their great collaborations and inimitable samples.

My dear fellow former and present students and colleagues, it was a pleasure to get to know you and work with you. Special gratitude goes to Dr. Chunxiang Yao, Dr.

Weidong Cui, Amanuel Kehasse, Dr. Nancy Leymarie, and Dr. Sandrine Voillard

Bourgoin. Dr. Jason Cournoyer, Dr. Cheng Zhao, Dr. Vera Ivleva, and Dr. David

Perlman thank you for your immense help during my initial stages of the program. Dr.

Konstantin Aizikov, Prof. Alexander Ivanov, Dr. Bogdan Budnik, and Prof. Eugene

Moskovets, thank you for your friendship and your willingness in help through all the years of my Ph.D. studies. Eugene, I truly appreciate your help well before and throughout the program; you are always ready with scientific advice and life guidance.

Most importantly, my wonderful family and friends, I’m so blessed to have all of you in my life. My beloved husband, Alexander Cherkassky, I would not be able to go through the obstacles, especially during the last months, without your endless help, moral support, and infinite love. I’m very lucky to deserve all the kindness and unconditional love of your wonderful family, especially, your Mother, Polina, and your sister, Rimma. To the best possible parents in life, my Mother, Alla, and my Father,

Pavel, and to my dearest sister, Olga - I never feel that we are across the ocean; you are always here with me in my heart. I can’t be thankful enough for you being there for me at all times – the day or the night.

DEAMIDATION AND RELATED PROBLEMS IN STRUCTURAL ANALYSIS OF

PEPTIDES AND PROTEINS

(Order No. )

NADEZDA P. SARGAEVA

Boston University School of Medicine, 2012

Major Professor: Peter B. O’Connor, Ph.D., Associate Professor of Biochemistry

ABSTRACT

Electron capture dissociation (ECD) and electron transfer dissociation (ETD) can generate unique fragments and preserve post-translational modifications (PTMs), enabling their detection in biological samples. They have been used to differentiate isomeric aspartic (Asp) and isoaspartic acids (isoAsp) produced upon non-enzymatic deamidation of asparagine (Asn) – a frequently occurring PTM. IsoAsp formation was detected in amyloid-β (A β) peptides in the specimens of Alzheimer’s disease (AD) patients, and is a potential biomarker for AD if it can be detected early in biofluids of live individuals. Synthetic isoAsp-containing A β fragments were studied using ECD to test the method's applicability. IsoAsp-7 and -23 were detected in top-down analysis of the

4.5kDa A β42 protein and in A β17-28 peptide. Further, a related method, electron ionization/impact dissociation (EID), was successfully applied to Asp/isoAsp differentiation for the first time. High-performance liquid chromatography (HPLC) is a powerful technique for the separation of complex mixtures. HPLC separation of Asp- and isoAsp-containing peptides revealed inconsistent elution orders, especially when isoAsp was located at the N-terminus, requiring ECD for identification. New diagnostic

fragments were proposed for N-terminal isoAsp based on the ECD and ETD results.

Challenges in detection of such fragments were improved by supplemental activation

and chemical modifications. Furthermore, a model for retention time prediction was

applied to isoAsp-containing peptides and suggested for their improved identification in

HPLC-MS/MS approach. IsoAsp is a β-amino acid, which distinctively contains an

additional methylene group in the backbone, forming a Cα-Cβ bond. Cleavage of this

bond provides diagnostic fragments for isoAsp by ECD. The same was proposed for

other β-amino acids. However, the Cα-Cβ bond cleavages were rare due to the instability

of the C β radical. Alternatively, in-source decay (ISD) fragmentation during matrix-

assisted laser desorption/ionization (MALDI) process can produce abundant ECD-like

fragmentation. It was proposed that use of hydrogen-accepting matrices may lead to

Cα-Cβ bond cleavage in β-amino acids, because the resulting radical would be stabilized by the carbonyl group. To test this, β-amino acid-containing peptides were analyzed by

MALDI-ISD using 5-nitrosalicylic acid matrix. The Cα-Cβ bond cleavages were observed.

Overall, new and improved methods were implemented allowing better characterization

and differentiation of β-amino acids.

vii

Table of contents

Title page…………………………………………………………………………………………..i

Reader’s approval page…………………………………………………………………………ii

Dedications……...... ……………………………………………………………………………. iii

Acknowledgements..…………………………………………………………………………….iv

Abstract…………………………………………………………………………………………...vi

Table of contents ...... viii

List of Tables ...... xiv

List of Figures ...... xv

List of Abbreviations ...... xx

Chapter 1 1

Introduction 1

1.1 Introduction to Mass Spectrometry in the Analysis of Biomolecules ...... 1

1.2 Introduction to Mass Spectrometry ...... 2

1.2.1 Ionization Techniques and Ionization Sources ...... 3

1.2.2 Mass Analyzers ...... 10

1.2.3 Tandem Mass Spectrometry ...... 29

1.2.4 ESI QqQ-FTICR mass spectrometer ...... 37

1.2.5 Bruker Daltonics solariX FTICR MS ...... 40

1.3 High Performance Liquid Chromatography (HPLC) ...... 42

1.3.1 Reversed Phase High Performance Liquid Chromatography (RP-HPLC) ...... 44

1.3.2 On-line and off-line Liquid Chromatography Mass Spectrometry ...... 48

1.4 Introduction to deamidation and isomerization of peptides and proteins ...... 49

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1.4.1 Deamidation and isomerization of peptides and proteins ...... 49

1.4.2 Biological importance of deamidation and isomerization ...... 51

1.5 Introduction to β-peptides ...... 53

1.6 Thesis overview ...... 56

Chapter 2 57

Identification of isoAsp in Amyloid βββ Peptides by Tandem ExD MS/MS 57

2.1 Introduction ...... 57

2.1.1 Amyloid β in Alzheimer’s disease...... 57

2.1.2 Isoaspartic acid formation ...... 58

2.1.3 IsoAsp in Alzheimer’s disease ...... 58

2.1.4 IsoAsp detection methods ...... 60

2.2 Experimental section ...... 62

2.2.1 Peptides and reagents ...... 62

2.2.2 Sample preparation ...... 62

2.2.3 Mass Spectrometry ...... 64

2.3 Results and Discussion ...... 64

2.3.1 Distinguishing the isomers ...... 64

2.3.2 ECD of the full length Amyloid beta protein fragment 1-40 ...... 67

2.3.3 Detecting isoAsp in Amyloid beta 1-42 ...... 69

2.4 Conclusion ...... 73

Chapter 3 74

The analysis of isoAsp, Asp, and Asn containing peptide variants by Liquid

Chromatography 74

3.1 Introduction ...... 74

3.2 Experimental section ...... 76

3.2.1 Peptides and reagents ...... 76

3.2.2 Liquid Chromatography ...... 76

3.2.3 Mass spectrometry ...... 77

3.2.4 RT prediction ...... 77

3.3 Results and Discussion ...... 78

3.3.1 RP-HPLC separations of isoAsp-peptides ...... 78

3.3.2 Predictive chromatography ...... 83

3.4 Conclusion ...... 86

Chapter 4 88

Differentiating N-terminal Asp and isoAsp residues in peptides 88

4.1 Introduction ...... 88

4.2 Experimental section ...... 89

4.2.1 Peptides and reagents ...... 89

4.2.2 Deamidation ...... 90

4.2.3 Chromatography ...... 90

4.2.4 Mass Spectrometry ...... 91

4.3 Results and Discussion ...... 92

4.3.1 Diagnostic fragments for the N-terminal Asp/isoAsp...... 92

4.3.2 Analysis of the deamidation products of Aβ1-10 (N1N7) by off-line RP-HPLC

followed by ECD ...... 94

4.3.3 Peak interference ...... 98

4.3.4 N-terminal acetylation ...... 98

4.3.5 Analysis of the N-terminal isoAsp by ETD ...... 100

4.3.6 The effect of ion activation ...... 102

4.4 Conclusions ...... 104

Chapter 5 105

Detection of IsoAsp in Amyloid β Peptides From CSF 105

5.1 Introduction ...... 105

5.2 Experimental section ...... 106

5.2.1 Standard peptides ...... 106

5.2.2 Immunoprecipitation of Amyloid β peptides from human CSF ...... 106

5.2.3 On-line nanoLC - ETD MS ...... 107

5.3 Results and Discussion ...... 108

5.3.1 NanoESI of Aβ standards ...... 108

5.3.2 On-line nanoLC - ETD MS analysis of the standards ...... 109

5.4 Future concepts ...... 112

Chapter 6 113

β-Peptide analysis by ExD tandem MS 113

6.1 Introduction ...... 113

6.2 Experimental section ...... 114

6.2.1 Peptides and reagents ...... 114

6.2.2 Sample preparation ...... 115

6.2.3 Mass Spectrometry ...... 115

6.3 Results and Discussion ...... 116

6.3.1 Q 06 beta peptide β2Vβ2Aβ2Lβ3Vβ3Aβ3L...... 116

6.3.2 Charge increase ...... 118

6.3.3 ETD of Q 06 beta peptide ...... 120

6.3.4 Modified Substance P ...... 122

6.3.5 Puma BH 3 protein analogues ...... 125

6.3.6 Proposed mechanism ...... 128

6.4 Conclusion ...... 134

Chapter 7 136

In-Source Decay for β-Peptide Analysis 136

7.1 Introduction ...... 136

7.2 Experimental section ...... 138

7.2.1 Peptides and reagents ...... 138

7.2.2 Sample preparation ...... 138

7.2.3 Mass Spectrometry ...... 138

7.3 Results and Discussion ...... 139

7.3.1 Diagnostic fragments for the C α-Cβ bond cleavage ...... 139

7.3.2 Advantages and disadvantages of the proposed method ...... 142

7.4 Conclusion ...... 143

Chapter 8 144

Conclusion and Future Perspective 144

xii

8.1 Conclusion ...... 144

8.2 Future Perspective ...... 147

Bibliography ...... 149

Curriculum vitae ...... 176

xiii

List of Tables

Table 3.1 The elution order results for peptides containing Asn, Asp, and isoAsp residues...... 82

Table 4.1 The effect of additional energy input on the diagnostic fragment formation.

Relative intensity (Rel. Int.) was calculated as the absolute intensity of the diagnostic fragment divided by the sum of the absolute intensities of all c and z fragments...... 102

xiv

List of Figures

Figure 1.1 The electrospray ionization process...... 6

Figure 1.2 The MALDI process...... 9

Figure 1.3 The schematic of a closed cylindrical ICR cell. Ions are trapped along the axis by the trapping potential applied to the trapping plates. Cyclotron motion of the ions is indicated, which is perpendicular to the magnetic field B...... 19

Figure 1.4 Ion motions in an ICR cell. The axis and the magnetic field face the paper.

Reproduced from Kolhinen, V. S.; et al, Commissioning of the double Penning trap system MLLTRAP, Nuclear Instruments & Methods in Physics Research Section a-

Accelerators Spectrometers Detectors and Associated Equipment , 2009, 600, 391-397, with permission...... 23

Figure 1.5 Excitation and detection of trapped ions in an ICR cell...... 24

Figure 1.6 A schematic representation of the orbitrap mass analyzer. The outer electrodes form the shape of a barrel (a). Ions ( i) move in spirals around the central spindle-shaped electrode ( b) while oscillating back and forth along the z axis...... 28

Figure 1.7 Peptide ion fragmentation nomenclature...... 31

Figure 1.8 The ESI qQq-FTICR mass spectrometer. Reproduced (modified) from

O'Connor, P. B. et al ., Rapid Communications in Mass Spectrometry, 20 , 259-266,

2006, with permission...... 38

Figure 1.9 The solariX TM FTICR mass spectrometer (Bruker Daltonics)...... 41

Figure 1.10 Isomerization of aspartic acid and deamidation of asparagine via a

succinimide intermediate...... 50

Figure 1.11 β-Peptide nomenclature...... 54

Figure 2.1 The ECD major pathway to produce c and z fragment ions (a); proposed ECD

mechanism for formation of diagnostic fragment ions (b)...... 63

Figure 2.2 ECD of A β17-28 (a), ECD of isoA β17-28 (b), and EID of isoA β17-28 (c)...... 65

Figure 2.3 ECD of A β40...... 69

Figure 2.4 ECD of A β42 deamidated at Asn7 and Asn27...... 72

Figure 3.1 IsoAsp position affects the peptide elution order...... 79

Figure 3.2 Chromatograms of Ang II peptide variants...... 80

Figure 3.3 N-terminal acetylation preserves the elution order of isoAsp modified peptides...... 81

Figure 3.4 Improved sequence identification (ID) ...... 84

Figure 3.5 Correlation between the predicted and the experimental retention time: Ang II variants separated in FA (a) experiment and (b) correlation; synthetic variants separated in TFA (c) experiment and (d) correlation...... 85

Figure 3.6 Improved identification of modified peptides...... 86

Figure 4.1 ECD spectra of the Angiotensin II (a) and its modified variant with isoAsp at

the N-terminus (b). Insets show side-chain losses from the charge-reduced species

+• +• [M + 2H] . IsoAsp specific fragment [M + 2H - 74] , produced as a result of the C α - Cβ

xvi

bond cleavage within the N-terminal isoAsp residue, is present in the modified variant

only, (b) inset...... 94

Figure 4.2 Chromatogram of the peptide mixture, generated by partial deamidation of the

Aβ1-10 (N1N7) peptide (top panel). Each chromatographic peak labeled based on ECD

results. ECD spectra shown correspond to the VIII-XI fractions with the isoAsp specific

fragments indicated in the insets...... 96

Figure 4.3. ECD spectra of chromatographic peaks I, IV-VII from the partially

deamidated A β1-10 (N1N7) peptide mixture...... 97

Figure 4.4 ECD spectra of the synthetic peptide variants with Asp (a) and isoAsp

residues (b) amidated at the C-terminus and acetylated at the N-terminus. IsoAsp1 and

+• • isoAsp5 specific fragments, [M + 2H - 116] and z6 - 57, are observed only for the isoAsp-peptide variant. Asp side-chain losses are also indicated, (a) inset...... 100

Figure 4.5 N-terminal isoAsp residue diagnostic fragment peak [M + 2H - 74] +• detection

for the A β1-10 peptide variant ( iD AEFRH iD SGY, fraction XI in the Figure 4.4, top panel)

by: (a) ETD on LTQ-Orbitrap, with supplemental activation energy parameter 15; and

(b) ECD on SolariX. Insets show diagnostic peaks and their S/N ratios...... 101

Figure 4.6 N-terminal isoAsp residue diagnostic fragment peak [M + 2H - 74] +• detection

using ETD without (a) or with SA (b) for the modified Ang II peptide variant

(iD RVYIHPF). Both spectra were acquired on Orbitrap. Insets show the S/N ratio of the

[M + 2H - 74] +• peak and the appearance of additional side-chain losses upon SA (His

and Tyr)...... 103

Figure 5.1 NanoESI mass spectrum of the A β40 standard peptide. Note the presence of

the A β26-40...... 109

xvii

Figure 5.2 LC-MS run of Aβ40, 200 fMol injected. On top is the total ion chromatogram

(TIC). In the middle is the base peak chromatogram for the 707.90-707.92 m/z region, corresponding to the doubly charged ions of Aβ26-40 peptide as confirmed by ETD, which is shown on the bottom...... 111

Figure 5.3 Hydrophobicity index of the Aβ40 peptide, generated by GPMAW 5.02...... 112

Figure 6.1 Q06 beta peptide β2Vβ2Aβ2Lβ3Vβ3Aβ3L: a) ESI, b) IRMPD, c) EID. Asterisk indicates noise peaks...... 118

Figure 6.2 ECD of Q06•cholamine...... 119

Figure 6.3 ETD of Q06 β2/β3-peptide...... 121

Figure 6.4 ECD of Substance P: a) unmodified, b) modified at Q5 and L10 to β3-type amino acids...... 123

Figure 6.5 ECD mechanism within α- vs. β-amino acid residues...... 124

Figure 6.6 ECD of Puma BH3 protein analogue I...... 126

Figure 6.7 ECD of Puma BH3 protein analogue II...... 127

Figure 6.8 ECD cleavage scheme of Puma BH3 protein analogues I and II...... 127

Figure 6.9 ECD mechanism for β-amino acid residues...... 129

Figure 6.10 ECD of triply charged substance P [M+3H]3+: a) unmodified, b) modified at

Q5 and L10 to β3-type amino acids...... 131

Figure 7.1 Formation of the ISD fragments: (a) c/z-type via hydrogen attachment and (b) a/x-type via hydrogen abstraction...... 137

xviii

Figure 7.2 MALDI-ISD-FTICR mass spectrum of modified substance Pusing 5-NSA

matrix. Data was acquired at the Bruker Daltonics facility, Billerica, MA...... 139

Figure 7.3 MALDI-ISD-FTICR mass spectrum of the β-Q06 peptide. Insets show the diagnostic ions for β2-valine-1, and β3-leucine-5 amino acids with corresponding region of a blank matrix as a control on top. * indicates abundant matrix cluster ions, ** indicates unidentified chemical noise peaks...... 140

Figure 7.4 Upon the loss of a hydrogen, amide nitrogen radical would cleave the Cα-Cβ

• bond producing diagnostic a-14 and x +14 in the case of the β3-type amino acid (a) and

• a-[side chain] and x +[side chain] in the case of the β2-type amino acid (b)...... 142

xix

List of Abbreviations

1,5-DAN 1,5-diaminonaphtalene

2,5-DHB 2,5-Dihydroxy benzoic acid

2-AA 2-aminobenzoic acid

2-AB 2-aminobenzamide

3D three dimensional

5-FSA 5-formylsalicylic acid

5-NSA 5-nitrosalicylic acid

Å angstroms

AA acetic acid

ABC Ammonium Bicarbonate

AC alternating current

ACN acetonitrile

AD Alzheimer's disease

AI-ECD activated-ion ECD

AI-ECD activated ion electron capture dissociation

Ala alanine

Ang II angiotensin II

APCI atmospheric pressure chemical ionization

APP amyloid precursor protein

APPI atmospheric pressure photoionization

Arg arginine

Asn asparagine

Asp aspartic acid

Aβ amyloid beta

BioLCCC liquid chromatography of biomacromolecules at critical

conditions

BUDA Boston University Data Analysis

CAD collisionally activated dissociation

CHCA α-Cyanohydroxy cinnamic acid

CI chemical ionization

CID collisionally induced dissociation

CSF cerebrospinal fluid

DC direct current

DE delayed ion extraction

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

ECD electron capture dissociation

EDD electron detachment dissociation

EI electron ionization

EID electron ionization dissociation

ESI electrospray ionization

ESI qQq-FTICR MS hybrid electrospray triple quadrupole FTICR mass spectrometer

ETD electron transfer dissociation eV electron volts

ExD electron activated dissociation

FA formic acid

xxi

FL Lorentz force

FTICR Fourier transform ion cyclotron resonance

FWHM full width at half maximum

GAGs glycosaminoglycans

GC/MS gas chromatography mass spectrometry

Gly glycine

HBTU hexafluorophosphate

HCAD high energy CAD hDM2 human oncogene product double minute 2

HFBA heptafluorobutyric acid

HILIC hydrophilic interaction liquid chromatography

His histidine

HOBt 1-Hydroxybenzotriazole hotECD hot electron capture dissociation

HPLC high performance liquid chromatography

ICR ion cyclotron resonance

ID inner diameter iD isoaspartic acid

ID identification

IEM inborn errors of metabolism

IgG immunoglobulin gamma

Ile isoleucine

IP immunoprecipitation

IRMPD infrared multiphoton dissociation

xxii

ISD in-source decay isoAsp isoaspartic acid isoD isoaspartic acid

IVR intramolecular vibrational energy redistribution kDa kilodalton keV kiloelectron volts

LC liquid chromatography

LC-MS liquid chromatography-mass spectrometry

Leu leucine m/z mass-to-charge ratio mAb monoclonal antibody

MALDI matrix assisted laser desorption ionization

MeOH methanol

Met methionine

MRM multiple reaction monitoring

MS mass spectrometry

MS/MS tandem mass spectrometry

MS2 tandem mass spectrometry

MSn tandem mass spectrometry nanoESI nanoelectrospray ionization nCI negative CI

NP-HPLC normal phase HPLC

OR orifice

PA picolinic acid

xxiii

PA protective antigen protein

PIMT protein L-isoaspartyl O-methyltransferase ppm parts per million

Pro proline

PSD post source decay

PTM post translational modifications

Q-o-TOF quadrupole orthogonal time-of-flight

QqQ triple quadrupole mass spectrometer

QTOF quadrupole – TOF

Rel. Int. relative intensity re-TOF reflectron-TOF

RF radio frequency

RGB Arg-Gly-isoAsp

RGN Arg-Gly-Asn

RNA ribonucleic acid

RNG ring electrode

ROP retinopathy of prematurity

RP reversed phase

RP-HPLC reversed phase high performance liquid chromatography

RT retention time

S/N signal to noise ratio

SA supplemental activation

SD smart decomposition

SEC size exclusion chromatography

xxiv

Ser serine

SK skimmer

SORI-CAD sustained off-resonance irradiation collision activated

dissociation

SRM selected reaction monitoring

ST stubbies

T Tesla

TEA triethylamine

TFA triflouroacetic acid

TOF time-of-flight

TOF/TOF time-of-flight/time-of-flight tandem mass spectrometer

TQMS triple quadrupole mass spectrometer

Trp tryptophan

Tyr tyrosine

UHV ultrahigh vacuum

UPLC ultra performance liquid chromatography

UV ultraviolet

UVPD ultraviolet photodissociation

xxv

Chapter 1

Introduction

1.1 Introduction to Mass Spectrometry in the Analysis of Biomolecules

Understanding the biological machinery of living organisms is the key to unveil the cause of and to cure and prevent diseases. Studying the structure and function of biomolecules can shed important light on life and disease. Mass spectrometry (MS) has become one of the most powerful tools to address this challenge in biomedical research because of its high sensitivity, specificity, speed and accuracy.

Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of the molecule. The history of MS began a century ago, when the first spectra of chemical elements or ions of single atoms and their isotopes were recorded. With advances of MS technologies, it is now possible to measure the molecular mass of metabolites, proteins and protein complexes, complex carbohydrates, oligonucleotides, lipids, and even whole viruses.

Biomolecules, such as proteins, appear to have distinct structures. The function, activity, and stability of a protein are determined by its structure, which is defined by its sequence, and possible post translational modifications (PTMs). Tandem mass spectrometry is a technique that allows gas-phase fragmentation of biomolecules such as proteins, providing accurate characterization of the protein structure. With tandem

MS, the protein sequence, type(s) of modifications as well as their locations within the molecule can be determined, thus allowing better understanding of its biological activity.

The power of MS was greatly enhanced when combined with liquid chromatography (LC), a technique that allows for separation of various molecules. LC -

MS is a very powerful method for quantitative and qualitative analysis of complex mixtures. It is routinely applied to analyze clinical samples for known disease biomarkers, as well as for new biomarker discovery in various biofluids.

In this thesis, ion-electron and ion-ion reaction based fragmentation techniques are used in tandem MS experiments to study two common post-translational modifications - asparagine deamidation and aspartic acid isomerization, as well as the related problem of β-peptide characterization. This chapter includes an introduction to

MS and LC. Various ionization methods, mass analyzers, fragmentation techniques, and LC separation methods are discussed, followed by a detailed description of the instruments used in this work. This chapter continues with an explanation of protein deamidation and isomerization, and concludes with an introduction to β-peptides.

1.2 Introduction to Mass Spectrometry

In a mass spectrometry experiment, analytes must be vaporized and ionized first, before they could be separated in electric and/or magnetic fields according to their mass-to-charge ratio ( m/z ), and detected. Therefore, all mass spectrometers contain the following major modules: an ionization source, a mass analyzer, and a detector. The mass analyzer and the detector could be a single unit as in the case of the Fourier transform ion cyclotron resonance (FTICR) MS. A fragmentation module may be added for tandem MS analysis performed tandem in space, which requires the use of two physically distinct mass analyzers. Alternatively, tandem MS analysis could be

performed in time inside of a single mass analyzer. This section will describe major

types of each module that are commonly used for biomedical research.

1.2.1 Ionization Techniques and Ionization Sources

Ionization refers to the process of ion production and an ionization source is a

mechanical mass spectrometer component where ionization takes place. The ionization

method/sources described below work by converting a neutral molecule into an ion

through electron capture, electron ejection, cationization, protonation, or deprotonation.

Conventional ionization techniques, such as electron ionization (EI), were limited

to small molecule analysis because of their requirement on sample volatility and thermal

stability. In EI, the harsh ionization condition (high energy electrons ~70 eV) may also

lead to extensive analyte fragmentation, which further limits its application to analysis of

large biomolecules. The development of new soft ionization techniques such as matrix

assisted laser desorption ionization (MALDI) 1-3 and electrospray ionization (ESI) 4-6 has made possible the analysis of large intact biological molecules such as peptides and proteins, which brought mass spectrometry to a revolutionary stage in biological research. Currently, MALDI and ESI are the preferred techniques for ionization of biomolecules, as these “soft” techniques possess the appropriate sensitivity, mass range and general performance for biomolecular mass spectrometric analysis.

Electron Ionization (EI)

Electron ionization (EI, formerly referred to as electron impact) is an ionization method in which high energy electrons interact with gas phase compounds to produce ions. 7 The sample for EI has to be delivered in a gas form, which is achieved by either

introducing the sample in a gas form via a capillary or “boiling off” liquid or solid sample

via thermal desorption. In many instances, before the sample is introduced into the

mass spectrometer via gas capillary, gas chromatography is utilized to provide the

desired analyte separation prior to MS detection (GC/MS). The typical ionization

mechanism involves irradiating analytes with 70 eV electrons, which eject an electron

from the molecule, leading to analyte ionization:

M + e - (fast) M+• + 2 e - (1.1)

Because an EI source employs thermal desorption of liquid and solid samples, followed by passing the sample through an electron beam, EI is considered a hard ionization technique. EI typically results in extensive fragmentation of precursor ions.

Additionally, the effectiveness of EI decreases for molecules above 350-400 MW because of the necessity of thermal desorption. The issues associated with the thermal desorption are typically manifested via the involatility of larger compounds, excessive fragmentation or thermal breakdown. 8

Chemical Ionization (CI)

In chemical ionization (CI), the ions are produced by colliding the analytes with

reagent gas ions that are present in the ion source via ion-molecule reaction. Some

commonly used reagent gases are: isobutene, methane and ammonia. Inside the CI

source, the reagent gas is present in large excess compared to the analyte. The

chemical ionization process starts with the ionization of reagent gas by electron impact.

This is followed by the reaction between reagent gas ions and reagent gas neutrals to

form an ionization plasma. The products of these ion-molecule reactions will react with

analyte molecules to generate analyte ions. The mechanism can be depicted as follows

(methane is used as an example of reagent gas):

- +• - CH 4 + e CH 4 + 2e (1.2)

+• + • CH 4 + CH 4 CH 5 + CH 3 (1.3)

+ + CH 5 + M MH + CH 4 (1.4)

Both positive and negative ions can be generated by reactions with this ionization plasma. Chemical ionization is a lower energy process than electron ionization, which yields less fragmentation, and usually generates a simpler mass spectrum. CI is more likely to produce the molecular ion than EI. 9

Electrospray Ionization (ESI)

Electrospray ionization (ESI) is an important mass spectrometry ionization

technique often used in biomolecular analysis. While first electrospray experiments

were performed in as early as 1930’s, it was not until 1989 when John Fenn’s work led

to the development of the modern day technique of electrospray mass spectrometry and

its applications to biomolecular analysis. 5 Fenn’s electrospray work was recognized with the Nobel Prize in Chemistry in 2002.

Electrospray ionization is a technique that produces gaseous molecular ions from a liquid solution under atmospheric pressure. 10 Analytes are dissolved in a solution that

allows for dissolution as well as easy evaporation (e.g. a 50:50 mixutre of methanol or

acetonitrile and water plus 0.1 % of formic acid). The solution is pumped through a

capillary, and a strong electric field (1-5 kV) is applied between the capillary and the MS

instrument inlet, often referred as the orifice. As a result, charge accumulates on the

surface of the liquid forming a “Taylor cone” at the tip of the capillary. 11 When charge

repulsion becomes stronger than liquid surface tension, highly charged droplets tear off

the cone peak. As a droplet travels towards the MS inlet, solvent molecules evaporate,

and the radius of the droplet shrinks and eventually reaches the Reyleigh limit, when

Coulombic repulsion exceeds surface tension, ultimately leading to “Coulombic

explosion” and formation of smaller droplets. Drying gas and heat are often applied to

help the solvent evaporation process to rid off the solvent molecules and other neutrals.

This process continues on until the unsolvated charged molecular ions are generated

and enter the mass spectrometer vacuum system. The process described above results

in the formation of positive ions and is illustrated in Figure 1.1. In order to generate and

detect negative ions, the polarity of the electric field is switched and a base instead of an

acid is added to the solvent.

Figure 1.1 The electrospray ionization process.

ESI is a very soft ionization technique that allows observing biologically native non-covalent interactions. 12 ESI often generates multiply-charged ions allowing analysis

of large molecules in a small m/z range (up to 3000). The presence of multiple charge

states for one molecule complicates the analysis of spectra, especially with complex

mixtures; fortunately modern software packages can mitigate this issue. ESI does not

suffer from matrix interference, but ion suppression can limit the analysis of complex

mixtures. ESI requires very clean samples and it is salt intolerant; thus, when analyzing

biological samples, a careful sample cleaning is required.

Nanoelectrospray ionization (nanoESI) is an electrospray ionization variant, where a capillary with a much smaller diameter is utilized and the sample is delivered without external force at a very low flow rate (nl/min vs. µl/min in ESI). The much lower flow rate becomes essential when analyzing biological samples with limited volumes.

Additionally, nanoESI generates much smaller droplets, thus reducing the necessary evaporation to generate ions. As a result, nanoESI is slightly more tolerant of salts and other impurities (less evaporation means that salt/impurities are concentrated down less during the nanoESI process).

Electrospray ionization is a powerful technique that is routinely used to analyze peptides, proteins, lipids, oligonucleotides, carbohydrates, various polymers and small molecules. ESI is a particularly powerful technique when on-line high performance liquid chromatography is coupled to a mass spectrometer providing analyte separation, sample clean up, and added specificity to biomolecular analysis.

Atmospheric-pressure chemical ionization (APCI)

Atmospheric-pressure chemical ionization (APCI) 9, 13 can be thought of as a

hybrid ionization technique between ESI and CI. Similar to ESI, sample in a liquid form

is delivered straight into the ionization source. However, the sample droplets are not

charged (as is the case for ESI), but are simply vaporized by heat application. Similar to

CI, solvent molecules are excited and ionized, followed by frequent collisions with the

analytes. Consequently, the analytes become charged via proton transfer or proton

loss, resulting in the formation of positive or negative ions, respectively. The differences

between APCI and CI are that APCI happens at atmospheric pressure and solvent

molecules are ionized by corona discharge rather than by electron irradiation.

APCI is amendable to on-line HPLC-MS systems to allow for analyte separation, sample cleanup and improved specificity. APCI typically generates singly charged ions due to its more energetic ionization process compared to ESI. APCI is particularly useful for mass analysis of non-polar compounds with moderate molecular weight up to

1.5 kDa.

Matrix Assisted Laser Desorption Ionization (MALDI)

Matrix Assisted Laser Desorption Ionization (MALDI), like ESI, is an important mass spectrometry ionization technique often used in biomolecular analysis. MALDI was first introduced through the works of Tanaka, Karas, Hillenkamp and colleagues. 1, 3

For his work, Tanaka shared the 2002 Nobel Prize in Chemistry with Fenn.

In order to perform MALDI mass spectrometry experiments, the analyte is first mixed with the matrix (typically weak organic acid, 10 2-10 6 times in excess) and crystallized on a metal plate. The surface of the crystal is then irradiated with a pulsed laser beam, which initiates matrix/sample ablation. As a result, the hot plasma plume of matrix ions and analyte neutrals is created. In this cloud of desorbed particles, multiple reactions can occur and the analyte molecules get ionized presumably via proton transfer from the protonated matrix ions (a.k.a. gas phase protonation model) or by preserving their charges from solution (a.k.a. lucky survivor model).14, 15 Once in the gas phase, the desorbed ions are guided by electrostatic forces into the mass analyzer. The ionization process described above is demonstrated in Figure 1.2.

Figure 1.2 The MALDI process.

MALDI sources typically employ ultraviolet (UV) lasers, such as nitrogen lasers

operated at 337 nm. 14 The matrix serves as an intermediate compound which indirectly

transfers the energy from the laser to the analyte, preserving it in a non-fragmented

state. This is the reason MALDI is also considered a soft ionization technique.

The matrix is often an easily crystallized small aromatic compound that readily

absorbs most of the photons, thus minimizing potential sample damage. There is a

great variety of matrices available for different types of analytes. 16 The most frequently used matrices for the analysis of peptide mixtures are α-Cyanohydroxy cinnamic acid

(CHCA) and 2,5-Dihydroxy benzoic acid (DHB), and sinapinic acid for proteins.

Since its introduction, MALDI has become a widespread ionization technique for analysis of peptides, proteins, oligonucleotides, carbohydrates, lipids, and other biomolecules. MALDI’s value for biomolecular analysis is manifested by its ability to generate molecular weight information for intact compounds. MALDI produces mostly singly charged protonated ions [M+H] +, but can also generate metal-adducted ions

[M+Me] n+ (Me = K, Na, Ca, and others) if small amount of salt is present in the sample

(MALDI can be tolerant to salts up to millimolar concentrations). MALDI is practical and

applicable for compounds with molecular weight up to 300,000 Da and provides

relatively simple spectra, which is advantageous for heterogeneous samples. Moreover, the pulsed nature of MALDI makes it well suited for the time-of-flight (TOF) mass analyzer.

MALDI has some disadvantages as well. One of its major drawbacks is the presence of abundant matrix clusters in the low m/z range of the spectra, which limits the analysis of molecules with m/z below 500-1000. Additionally, the possibility of photo- degradation of analytes of interests by laser ablation exists. Finally, singly charged ions that are generated by MALDI might preclude certain tandem MS methods, such as those requiring multiply charged precursor ions ( vide infra ).

1.2.2 Mass Analyzers

The ionized analytes can be separated according to their m/z as they now can be manipulated by the electrical and magnetic field. A mass spectrum can then be recorded to provide the m/z information to the user. The first MS instrument was called a mass spectrograph, because the spectrum of the m/z values was recorded as a photograph. When the mass measurement was recorded as electronic signal, the instrument's name became the mass spectrometer.

There are many types of mass analyzers available today. They differ by their principles of operation and the following important properties: the mass range limit, the dynamic range, the mass accuracy, the resolving power (a.k.a. the resolution), the sensitivity, the scanning speed, tandem MS analysis capabilities, and cost. These characteristics are described below and throughout each subsection within this chapter.

The mass range limit is the m/z range between the low and the upper thresholds that could be measured by the mass analyzer.

The dynamic range is a measure of the detection range of a detector, which is characterized by the ratio of the largest to the smallest detectable signals (related to the number of ions that can be detected).

The mass accuracy is the ratio of the experimental m/z error to the theoretical

m/z value, which is usually measured in parts per million (ppm). Mass accuracy is linked

to the resolving power of the analyzer: low resolving power analyzers cannot provide

high mass accuracy.

The resolving power , or resolution, is the ability of a mass analyzer to distinguish

signals for two distinct ions with close m/z values. It can be calculated as

R = M/ ∆M (1.5)

where,

M = (( m/z )1 + (m/z )2)/2 (1.6)

∆M = |( m/z )1 - (m/z )2| (1.7)

The resolving power can also be determined with a single peak, where ∆M is its

full width at half maximum (FWHM).

The mass spectrometer's sensitivity is the lowest amount of sample that could be

detected. In the ideal case, it would a single ion. The sensitivity depends on the

efficiency of ion transmission though the mass spectrometer, defined as the ratio of the

number of ions reaching the detector to the number of ions generated in the ion source.

The scan speed is the rate at which the analyzer measures over a particular mass range.

Tandem mass spectrometric analysis refers to the process where specific ions

(often referred to as parent ions or precursor ions) were separated and selected for fragmentation (via collision with inert gas or by other techniques) to generate secondary

ions (often referred to as product ions, fragment ions, or daughter ions) whose m/z values are then measured. Tandem mass analysis provides additional specificity to the measurement of unknown analytes. Tandem mass spectrometry is described in more details in Chapter 1.2.3.

The cost of the instrument depends on the combination of various modules which comprise the mass spectrometer.

Quadrupole Mass Analyzer

Quadrupole mass analyzers have been utilized with EI sources since 1950’s and are the most widespread mass analyzers today. 9 Since late 1980’s and early 1990’s,

quadrupole analyzers have been utilized with ESI and APCI ionization sources. There

are a few factors that led to quadrupole analyzers being the most common mass

analyzers. One of these factors is their low cost. Secondly, quadrupole analyzers can

operate at a relatively high vacuum pressure, which makes them compatible with high

flow-rate liquid-based ionization sources, such as ESI and APCI. Additionally,

quadrupole mass analyzers possess wide m/z range of up to 4 kDa.

The quadrupole analyzer, as its name implies, consists of four parallel metal

rods. Ions are constrained within the boundary of the quadrupole under influence of a

combined direct current (DC) and radio frequency (RF) potentials applied to the rods.

This spatial quadrupole arrangement generates an oscillating electric field, which only

allows ions with specific m/z to travel the entire length of the quadrupole mass analyzer,

whereas other ions are filtered out.

Quadrupole mass analyzers have poor mass accuracy of ~100 ppm, limited

resolution of ~4000, wide mass range of up to 4000 m/z , and slow scan speed (about a

second for the entire mass range).

Quadrupole mass analyzers are amendable to performing tandem mass spectrometry experiments when three such analyzers are placed sequentially. Such a tandem mass spectrometer instrument is referred to as a triple quadrupole MS (QqQ, or

TQMS). The first quadrupole, Q1, scans and selects the ion of interest (a.k.a. the parent or precursor ion). The second quadrupole, q2, or the collision cell, is where the selected parent ion is transmitted to and undergoes collisions with an inert gas (e.g.

Argon) to induce collisionally activated dissociation (Chapter 1.2.3). The third quadrupole, Q3, is utilized to scan and analyze the resultant fragment ion(s). ESI/APCI

QqQ instruments are the mass spectrometers of choice for small- and medium-sized analyte quantification in biomedical and in vitro diagnostics fields. Isotope dilution mass spectrometry technique is often used with these instruments for accurate quantitation of various biomarkers in order to screen for or to diagnose various disorders in clinical diagnostics laboratories.

Quadrupole Ion Trap Mass Analyzer

Ion trap mass analyzer was invented by Wolfgang Paul who later received the

Nobel Prize in Physics in 1989 for this work. Following the invention of the ion trap analyzer, work by Stafford et al. resulted in the first commercially available ion trap mass analyzers. 17 A quadrupole ion trap operates on the similar principle as a quadrupole mass analyzer; however, the ions are trapped in the ion trap mass analyzer instead of passing through it as in the quadrupole mass analyzer. An ion trap consists of two hyperbolic endcap electrodes and a ring electrode in between the endcap electrodes. Ions are trapped in this three dimensional (3D) space by applying oscillating

RF voltages to the ring electrode and non-oscillating, static DC voltage to the end cap electrodes to create a 3D quadrupolar electric field. By changing the radio frequency of

the auxiliary alternating current (AC) electric field applied to the end cap electrodes, the

ions are ejected through a small hole in the endcap electrode and towards the

detector. 17 In addition to 3D ion traps, there are also linear ion traps. A linear ion trap utilizes the quadrupole arrangement with sectioned quadrupoles, and uses two dimensional RF field to trap the ions. 18 A linear ion trap can be used as a selective ion

filter or an actual ion trap. Linear ion traps have higher ion capacity than 3D traps, thus

higher dynamic range.

Ion traps have very similar performance to quadrupole mass analyzers. Ion traps

have poor mass accuracy of ~100 pm, low resolution of ~4000, wide mass range of up

to 4000 m/z , and slow scan speed (about a second for the entire mass range). Similar

to quadrupoles, ion traps are low-cost instruments. Ion traps in theory have higher

analytical sensitivity than quadruples due to their ability to accumulate ions.

Ion traps are also amendable to tandem mass spectrometry, but it is not

performed tandem in space (as is the case with quadrupoles), but in time. This is

achieved because of the ability of an ion trap to select a single ion species of interest,

while ejecting all other ions, with gas pulsing into the ion trap chamber to facilitate

collisions between the selected ions and gas molecules, which subsequently results in

formation of fragment ions. Moreover, the resultant fragment ions can be isolated by the

same process as described above, and further fragmented to generate secondary

fragment ions. This process of multiple tandem mass spectrometry analysis is known as

the MS n. Additionally, ion traps can be used to perform tandem mass analysis by electron transfer dissociation (ETD) to generate complementary c and z fragment ions. 19

This technique will be described later.

Ion traps with ESI or MALDI sources are utilized for a number of biomolecular

applications such as protein characterization by MS and MS n analysis of proteolytic

digests, carbohydrate characterization and analysis as well as small molecule analysis.

Time-of-Flight Mass Analyzer (TOF)

A time-of-flight (TOF) mass analyzer is the simplest analyzer based on its

physical design. The origin of TOF analyzers goes back to 1946 through the work of

Stephens. 20 In a TOF mass analyzer, a cluster of different ions is accelerated through a

flight tube at 10 -7 Torr vacuum by an electric field of a constant, known strength.

Because the accelerating potential is constant, all ions have the same kinetic energy

when they exit the source; however, lighter ions will have higher velocities and thus

reach the detector first, whereas heavier ions will reach the detector at later times.

Therefore, as its name implies, a TOF analyzer measures the time it takes for an ion to

reach the detector, which is subsequently converted to its m/z value.

Simple linear TOF analyzers such as one described above, have reasonable performance characteristics, but have been enhanced over the years to perform modern day biomolecular mass analysis. Linear TOFs have infinite m/z range in theory, poor mass accuracy of ~200 ppm, moderate mass resolution of ~8000 and fast scan speed

(in the millisecond scale). These analyzers are generally of low cost and cannot be utilized for tandem mass spectrometry analysis. More recently, TOF analyzers have been improved to deliver much better performance for biomolecular analysis.

Specifically, reflectron-TOF (re-TOF) and delayed ion extraction (DE) for MALDI sources have been at the forefront of the TOF performance enhancement.

Early MALDI-TOF instruments suffered from poor mass resolution and subsequently poor mass accuracy. This was due to the fact that ions were accelerated

instantaneously in the MALDI source into the flight tube. This resulted in ions with the

same mass hitting the detector at slightly different times due to the fact that ion plasma

plume formation by laser ablation and desorption is not an instantaneous, but rather a

timed process lasting a finite time period, leading to the temporal spread of the originally

formed ion packet. Moreover, the ion kinetic energy spread resulted in further

degradation of the TOF resolution. This shortcoming of MALDI-TOF systems was

overcome by an innovation known as the delayed extraction. 21 DE works by allowing for cooling and focusing of the ions prior to ion acceleration, which takes about 200 nanoseconds, thus the delayed extraction. Physically, this is achieved by pulsating high voltage to the extraction plate (between the MALDI target plate and the drift tube entrance) on a nanosecond scale in unison with MALDI laser pulses. This causes the ions that are further from the drift tube entrance being accelerated more towards the entrance and the ions that are closer to the entrance being accelerated less. In turn, the net result is an ion packet with narrower kinetic energy distribution prior to their entrance into the flight tube, ultimately resulting in tighter temporal and spatial ion distribution hitting the detector, thus the increased resolution.

Another important innovation that led to TOF being a major tool for biomolecular analysis was re-TOF. 22 Re-TOF combines the time-of-flight concept with an electrostatic

mirror. The purpose of the electrostatic mirror is to reflect (hence the name) the ions

towards the ion detector while improving the mass resolution. The electrostatic field

allows separation of the ions that have the same m/z ratio, yet different kinetic energies,

by increasing the amount of time it takes ions to reach the detector: higher energy ions

penetrate more deeply into the reflectron, thus spending more time inside the reflectron,

which partially offset their shorter flight time outside of the reflectron. Therefore, re-TOF

improves the mass resolution by improving the kinetic energy focusing and by increasing

the ion path length; however, the benefit of re-TOF drops off above 5,000 m/z . Re-TOFs have a mass range up to 10,000 m/z , are fairly accurate at ~10 ppm or better, with a high mass resolution of ~15,000 or much better (e.g. the latest commercial Bruker

Daltonics ultrafleXtreme TOF has a claimed 1 ppm mass accuracy and 40,000 mass resolution) and fast scan speed in the millisecond scale. These analyzers are moderately priced and can be utilized for tandem mass spectrometry analysis. Further, modern day TOF mass spectrometers can utilize not only MALDI sources, but also ESI and nano-ESI sources.

Tandem mass spectrometry experiments are possible with re-TOF mass analyzer via a technique known as the Post Source Decay (PSD). PSD is a process in which a parent ion dissociates into fragment ions after it leaves the ionization source and travels through the flight tube. Because PSD occurs in the flight tube where the ion velocity has already been established, the parent and the fragment ion(s) have the same velocity, and would arrive at the detector at the same time in linear TOF instruments, making them indistinguishable from each other. In re-TOF instruments, however, parent and fragment ion(s) have different kinetic energies, and will penetrate the reflectron to different depths, thus producing the parent and fragment ion spectra. Alternatively, fragmentation can take place inside the ion source, a process known as the in-source decay (ISD), which will be described in details later.

There are two tandem mass spectrometers utilizing TOF mass analyzers: the quadrupole – TOF (QTOF) and the TOF/TOF instruments. QTOF tandem mass spectrometry is conceptually similar to QqQ described above. The first mass analyzer, a quadrupole, scans, selects and guides the parent ions into a collision cell. The resultant

product ions are then analyzed by a TOF reflectron mass analyzer. A QTOF instrument

can provide much higher mass accuracy and mass resolution than a QqQ instrument.

The MALDI-TOF/TOF instrument is another tandem mass spectrometer utilized

for biomolecular analysis. 23 For these types of experiments, the first TOF analyzer (with

a linear flight tube) is used to select the parent ions, and the second TOF analyzer is a

reflectron used to detect and analyze the fragment ions. The generation of fragment

ions is facilitated by PSD, by colliding with gas molecules in the collision cell, and by

ultraviolet laser irradiation. 24

Fourier Transform - Ion Cyclotron Resonance Mass Analyzer

The Fourier transform - ion cyclotron resonance (FTICR) mass analyzer was first introduced for the mass spectrometry analysis in 1974 by Comisarow and Marshall. 25

Among all different types of mass analyzers, the FTICR is capable of achieving the

highest performance in terms of resolving power and mass accuracy, yet it is a

complicated and an expensive system. The FTICR mass analyzer is an electromagnetic

trap, known as the Penning trap, which uses a combination of an electric and a magnetic

field to trap ions inside the ICR cell.

The ICR cell . The ICR cell is one of the main components of an FTICR mass

analyzer, where mass analysis takes place. It is positioned inside a homogeneous

magnetic field. Many different geometries of ICR cells have been implemented over

years, including the cubic cell, the Infinity cell, the open and closed cylindrical cells, the

three-sectioned cell, cell with hyperbolic electrodes, cell with hemisphere end caps, dual

cells, the elongated cell, and others. 26, 27 One example cell geometry is schematically shown in Figure 1.3. This is a closed cylindrical ICR cell. The cylinder consists of two pairs of electrodes – one for ion excitation and the other for detection of the image

current . End cap electrodes are used to trap ions along the magnetic field axis. The principles of ion motion and detection are explained below.

Figure 1.3 The schematic of a closed cylindrical ICR cell. Ions are trapped along the axis by the trapping potential applied to the trapping plates. Cy clotron motion of the ions is indicated, which is perpendicular to the magnetic field B.

The magnet . Magnetic field is provided by an electromagnet, which is a main component of an FTICR mass analyzer. An electromagnet is made from a coil of wire that acts as a magnet when an electric current passes through it. Magnets used in

FTICR-MS are primarily superconducting magnets made of superconducting wire.

These magnets can produce greater magnetic fields than non-superconducting electromagnets. The magnetic field strength in an FTICR mass spectrometer typically ranges from 4 Tesla (T) to 18 T. An FTICR MS with a 21 T magnet is currently under development at the National High Magnetic Field Laboratory and PNNL.28 An increase in the magnetic field strength improves the resolving power, signal-to-noise ratio, and mass accuracy of the mass spectrometer. However, the cost of the mass spectrometer also increases with the magnetic field strength, e.g. a 14.5 T FTICR magnet cost is approximately $2,000,000.

Ion transport . Ions can be generated inside or outside the ICR cell. Traditionally,

EI and CI were used to generate ions inside the ICR cell. For analysis of large biomolecules such as proteins, however, soft ionization techniques, mostly ESI and

MALDI, are usually used as external ionization sources. Once generated in the external ion source under atmospheric pressure or at relatively high pressure, ions need to be transferred to the ICR cell which operates in ultrahigh vacuum (UHV). Therefore, several differential pumping stages are usually needed to maintain the pressure difference of several orders of magnitude between the ion source and the mass analyzer. Ion transfers from the ionization source to the ICR cell can be achieved with implementation of various ion optics, such as ion funnels, RF-multipole ion guides, electrostatic lenses and various focusing lenses.

Ion motion . Inside the magnetic field, an ion with mass m, charge q, and velocity v experiences the Lorentz force ( FL) and circles an orbit of radius r in a plane

perpendicular to the direction of the magnetic field B:

FL = qE + q (v x B) (1.8)

where E is the electric field. Ideally, in the center of the cell ion motion is

unaffected by the electric field, thus:

FL = q (v x B) (1.9)

The ion trajectory with velocity v moving in the magnetic field B is stable if the

centripetal force (the inward force) and the centrifugal force (the outward force) are

equal:

(mv 2)/r = q (v x B) (1.10)

The angular velocity

ω = 2πr = v/r = (qB)/m (1.11)

Equation (1.11) is called the cyclotron equation and ω is the cyclotron frequency .

The cyclotron frequency of an ion is inverselyproportional to its mass to charge ratio in a

constant magnetic field and is independent of the ion velocity. This is the principle of

mass analysis in FTICR MS, and ω is used to calculate the ion m/z ratio (where z is q/e ,

and e is the elemental charge).

Ion motion described in Equation (1.11) is called the cyclotron motion, which is

confined to a plane perpendicular to the magnetic field direction. Ion trapping along the

magnetic field lines is achieved by applying a small potential (usually in the order of ~1 V

(Vtrap )) to the end cap electrodes of the same polarity as the ion charge. As a result, ions

oscillate along the z-axis of the ICR cell. The potential applied to the end cap electrodes

also creates a repulsive electric field in the xy-plane, which pushes ions towards the

excitation and detection plates. The interplay between ions and the radial electric field

and the axial magnetic field induces a third motion of ions, the magnetron motion, which

causes the center of the ion cyclotron orbit to circle around the ICR cell axis.

The radial force Fradial , which ions experience due to the radial electric field induced by trapping potential Vtrap , opposes the inward-directed Lorentz magnetic force

FL from the applied magnetic field and can be described as:

2 Fradial = qE(r) = (qV trap α r) / a (1.12)

where a is the distance between the trapping plates, and α is a cell geometry factor. The Lorentz force equation (1.10) for the plane perpendicular to the magnetic field Bz can be reduced to:

2 mω r = qB zωr (1.13)

To obtain the equation for ion motion one needs to consider the total force

combining equations (1.12) and (1.13):

2 2 mω r = qB zωr - (qV trap α r) / a

2 2 or ω - (qB zω)/m + (qV trap α) / a m = 0 (1.14)

Equation (1.14) is a quadratic equation, with two solutions:

2 2 1/2 ω+ = ωc/2 + ( (ωc/2) – (ωz /2) ) (1.15)

2 2 1/2 ω- = ωc/2 - ( (ωc/2) – (ωz /2) ) (1.16)

where ω+ and ω- are the reduced cyclotron and magnetron frequencies, in which

ωc is the unperturbed cyclotron frequency, and ωz is the trapping oscillation frequency of

the ions moving in the z-direction, defined as (from (1.12)):

2 1/2 ωz = ((2qV trap α r) / ma ) (1.17)

The magnetron motion causes radial diffusion of the ion and reduces its cyclotron

frequency. The ωz is proportional to the trapping potential, introducing a trade-off between efficient trapping of the ions and perturbing the cyclotron frequency. For this reason Vtrap is usually kept low. Magnetron and axial frequencies are usually much less

than the cyclotron frequency, and generally are not detected. An ion trajectory that

shows all three motions experienced by the ion in an ICR cell is demonstrated in Figure

1.4.

Figure 1.4 Ion motions in an ICR cell. The axis and the magnetic field face the paper .

Reproduced from Kolhinen, V. S.; et al, Commissioning of the double Penning trap

system MLLTRAP, Nuclear Instruments & Methods in Physics Research Section a -

Accelerators Spectrometers Detectors and Associated Equipment , 2009, 600, 391-397 ,

with permission.

Ion excitation and detection . Since the cyclotron frequency is constant for an ion

of a given m/z , the radius of an ion's cyclotron orbit depends on its velocity or its kinetic energy. At room temperature, in a magnetic field of 9.4 T, the thermal cyclotron radius of a singly charged 1 kDa ion is rather small, ~0.08 mm. 27, 29 In order for ions to be

detected, their cyclotron orbit radii need to be increased, so that ions circle at higher

orbits close to the cell electrodes, which are typically ~5 cm away from the center. The

coherent excitation of ions is achieved by differentially applying an RF voltage sweep

containing a band of frequencies corresponding to the desired m/z range to excitation electrodes (called a chirp). 27 Each ion will respond only to its particular RF pulse corresponding to its cyclotron frequency. As the ion absorbs the RF energy, it is resonantly accelerated, increasing its cyclotron radius to that of a higher orbit (Figure

1.5). This process is called the Ion Cyclotron Resonance excitation, which gave rise to the name of the ICR cell. The radius of excited ions is dependent on the amplitude of the electric field E0 and the duration of the resonant excitation T for a given magnetic field B, but is m/z independent, so all ions accelerate to the same orbit, provided that the excitation amplitude and duration are constant across the whole m/z range: 30

r = (E 0T)/2B (1.18)

When the ion packet moves to the proximity of a detection plate, it induces accumulation of charges with opposite signs to the ion packet at the plate (image charges). As ions rotate around the center of the ICR cell, image charges are induced alternately at opposite detection plates, creating an alternating image current between them. This image current is converted to an alternating voltage, which is amplified to generate a time domain signal (a transient). The acquired transient contains the cyclotron frequency information of all ion packets, which can be recovered via a mathematical operation called the Fourier transform. The frequency domain spectrum is then converted to the mass spectrum via the cyclotron equation.

Figure 1.5 Excitation and detection of trapped ions in an ICR cell.

Ions excited to a lager orbit will eventually lose their energy and spiral back down

to the center of the cell, so the signal on detection plates will drop over time. In addition,

ion collisions with the background gas will cause the loss of coherence of the oscillating

ion packet, dispersing it into a donut-shape. This in turn causes the loss of the

differential signal and a fast decay of the transient. Thus, the pressure of the ICR cell

should be kept below 10 -8 torr (ideally10 -9 torr or lower) to reduce collisions during the

transient acquisition. The duration of the transient T is important as it directly influences

the FTICR resolving power R:31

R = (f T)/2 (1.19)

FTICR MS performance . From the equation (1.19), it is apparent that the

resolving power is also linearly dependent on the strength of the magnetic field. Thus,

the magnet field strength and a good vacuum are essential for achieving the excellent

performance of the FTICR MS, although its performance is also influenced by many

other factors, such as the electric and magnetic field homogeneity, the cell geometry, ion

transmission, and electronics, etc. The typical mass resolution achieved on a 12 T

FTICR instrument is over 200,000 at m/z 400 in the analysis of proteins. Because ions can be trapped in the ICR cell for minutes or even hours, mass resolution greater than

1,000,000 can also be obtained. The typical mass accuracy is in the range of 1 ppm or better when internally calibrated, and in the low ppm range when externally calibrated for peptides of m/z ~1000-2000. The usual m/z range is 150-3,000, but can be extended up to 5,000 to detect ions of a mass of ~100 kDa. The low-mass cutoff is primarily limited by the sampling frequency to satisfy the Nyquist limit, but could also result from resonant excitation of ions by the multipole RF field as ions traverse the magnetic field gradient.

For commercial FTICR instruments, the upper mass limit is primarily determined by the

ion transfer optics. The routinely achievable sensitivity of an FTICR MS is in the fmol

range. The ICR dynamic range and the upper mass limit scale with the strength of the

magnet field, and analysis/experiments which are difficult or impossible with a weak

magnet can be routinely performed with a stronger magnet. When combined with high-

resolution LC separations, the overall dynamic range of measurements can exceed

10 6.32 The FTICR MS scan speed is compatible with the LC-MS time scale (in seconds); however, to achieve the best resolving power, longer transient acquisition is required for a given magnetic field strength. Use of a magnet with a higherfield strength can also improve the scan speed.

Applications . A unique FTICR MS feature is its ability to perform tandem MS

inside the ICR cell. Multiple tandem MS methods can be implemented in an FTICR

instrument. In particular, up to recently, electron capture dissociation (ECD) 33 was only

available in FTICR MS. Other tandem MS methods suitable for FTICR MS include

sustained off-resonance irradiation collision activated dissociation (SORI-CAD), 34 and infrared multiphoton dissociation (IRMPD) 35 , etc.29, 36 The versatile tandem MS capability

afforded by an FTICR MS, in addition to its ultra high resolution and mass accuracy, and

coupling to liquid chromatography led to its wide applications in both top-down and

bottom-up analysis in proteomics, including protein sequencing, PTM characterizations,

study of protein conformations, and analysis of protein complexes. 37 FTICR MS is used

to analyze complex chemical mixtures such as crude oil, which can be made up of tens

of thousands of different types of molecules. It is also used to determine the molecular

structure of carbohydrates, nucleic acids and other biomolecules. In addition, FTICR MS

can identify the chemical fingerprint of explosives and accelerants, thus, can be used for

counterterrorism efforts. Overall, FTICR MS allows obtaining such information on the

sample as its elemental composition and its associated class, type, and carbon

distribution information, amino acid composition, metal ion oxidation state, charge state

distribution, identification of functional groups, isotopic distributions, and ultimately,

isotopic fine structure. 38

Orbitrap Mass Analyzer

The orbitrap is an electrostatic ion trap where ions are trapped by an electrostatic field. 39 The technology is based on the Kingdon ion trap. 40 The orbitrap mass spectrometer was recently introduced by Makarov and has been widely used in biological research since. 41, 42 The orbitrap operates by radially trapping ions about a

central spindle electrode (Figure 1.6). Ions move in spirals around the central electrode.

The axial component of the trapping field contains a quadratic term of the axial

displacement, and the resulting restoring force is proportional to the axial displacement.

Consequently, the ion axial motion takes the form of a harmonic oscillator, with its

frequency inversely proportional to the square root of the ion m/z value. The outer

electrode is split in the center by insulating ceramic ring and is used to detect the image

current induced by the moving ions along the axis. The resultant transient is digitized

and Fourier transformed to obtain the axial frequencies of ion oscillations, which are then

converted into m/z values using the mass calibration equation to obtain the mass spectra.

Commercially available orbitrap instruments feature ESI, nanoESI, and APPI ionization sources. The ion source is connected to the orbitrap via a linear ion trap capable of generating MS and MS n spectra itself, and a C-trap. 42 The C-trap is a bent

quadrupole named after its C-shape. The C-trap allows ion accumulation, concentration

into a small cloud, and radial injection into the orbitrap. The injection is accommodated

by focusing electrodes and a repeller at the orbitrap entrance. The central electrode

voltage is lowered at the time of ion injection to allow ion entrance into the orbitrap.

r a z

Figure 1.6 A schematic representation of the orbitrap mass analyzer. The outer

electrodes form the shape of a barrel (a). Ions ( i) move in spirals around the cent ral

spindle-shaped electrode ( b) while oscillating back and forth along the z axis.

The orbitrap instrument performance is quite different from that of quadrupole ion traps. Compared with a Q-TOF instrument, the resolution is dramatically increased, and so is the dynamic range. The mass resolving power of the first-generation commercial orbitrap at m/z 400 with a 1 second transient is >60,000, with its maximal resolving power exceeding 100,000. At low m/z values, the orbitrap resolution is typically less

than that obtainable on an FTICR instrument. However, the resolution of an orbitrap

decreases not as rapidly as that of an FTICR when the m/z increases. The mass accuracy of an orbitrap is <5 ppm with external calibration, and <2 ppm with internal calibration. The instrument sensitivity is at the sub-fmol level with dynamic range greater than 10 3. The mass range is limited by the use of the linear ion trap and is either m/z 50-

2,000 or m/z 200-4,000. It is possible to detect 4 scans per second, acquiring 1 high resolution MS scan in the orbitrap and the rest in the LTQ as MS/MS scans. The latest improvements of the system with the implementation of the high-field orbitrap and advanced signal processing allowed further increase in its analytical performance. 43 An

average mass resolution of 220,000 was achieved in the analysis of proteins. 44 Thus, the resolving power has become comparable with that obtained with 7 Tesla FTICR mass spectrometers. It is now possible to perform LC-MS/MS experiments on intact proteins of up to 50 kDa molecular weight. 45

The orbitrap's ability to perform on the LC time scale has allowed its application

to both small molecule analysis, such as metabolomics and metabolite analysis, drug

metabolism, doping control and food contaminants, and proteomics. 46, 47 Implementation

of the electron transfer dissociation (ETD) on the orbitrap further expanded its

application in proteomics research. 48 Combining three different and complementary fragmentation techniques - CAD, high energy CAD (HCAD), and ETD, the orbitrap is capable of characterizing labile PTMs, such as phosphorylation and glycosylation. It has been used for the differentiation of phosphorylation and sulfation, glycan structure elucidation, PTM site determination, sequencing of peptides, top-down and middle-down analyses, and protein quantitation via stable isotope labeling.

1.2.3 Tandem Mass Spectrometry

Tandem mass spectrometry (MS/MS, MS 2 or MS n) involves multiple stages of

mass analysis with ion fragmentation happening between these stages. Tandem mass

spectrometry is a three-step process: isolation of a precursor or parent ion of interest

(first stage of mass analysis), fragmentation of the parent ion, and detection of the

resultant fragment ions (a.k.a. daughter or product ions) during the second stage of

mass analysis. These mass analysis events might be happening in different mass

analyzers or in the same mass analyzer. The former is typically referred to as “tandem

mass spectrometry in space” due to the fact that mass analyses happen in distinct

physical locations. Examples of mass spectrometers implementing tandem-in-space

mass analysis are QqQ, QTOF, TOF/TOF, and others. The latter is known as “tandem

mass spectrometry in time”, in which isolation of the parent ion, its fragmentation and the

subsequent detection of the product ions are happening in the same mass analyzer, yet

at different times. Examples of mass spectrometers capable of performing "tandem-in-

time" mass analyses are 3D quadrupole and linear ion traps, and FTICR MS. As

described above, tandem-in-time allows for MS n analysis.

There are a number of different tandem mass spectrometry experiments or modes. These are:

Product ion scan – in this mode, the first mass analysis event selects the precursor ion of interest, which is fragmented followed by detection of all product ions in the second mass analysis event. This tandem mass analysis experiment can be performed on both in-space and in-time instruments.

Precursor ion scan – in this mode, the second mass analyzer detects a product ion of interest whereas the first mass analyzer scans for precursor ions that can produce this product ion. This mode can only be performed on in-space instruments.

Neutral loss scan – in this mode, both the first and the second analyzers scan

across specified mass ranges simultaneously. The difference in the scanned masses for

the analyzers matches the mass of the neutral loss of interest. Neutral loss scan can

only be performed on in-space instruments.

Selected reaction monitoring (SRM) – the first and the second mass analyzers are set to detect specific precursor and product ion masses, respectively. Multiple reaction monitoring (MRM) is a mode in which multiple SRMs are cycled. SRM experiments are usually performed on in-space instruments.

Figure 1.7 Peptide ion fragmentation nomenclature.

All these tandem mass spectrometry modes can be used to help deciphering the structure of or to quantitate various biomolecules. For protein and peptide mass analysis specifically, these modes are the necessary tools to deduce the structure or the sequence of the peptide/protein of interest. A common nomenclature describing peptide/protein product ions was proposed in 1984 by Roepstorff et al .49 This ion

fragmentation nomenclature is demonstrated in Figure 1.7. For a peptide containing m amino acid residues, fragmentation between the nth and the ( n+1) th amino acids would

provide N-terminal fragment ions an, bn, and cn, and C-terminal fragment ions xm-n, ym-n,

zm-n, depending on the bond cleaved. The identity of an amino acid residue can be deduced (with the exception of isomeric Leu/Ile residues) from the mass difference of fragment ions of the same series resulting from bond cleavages immediately N-terminal and C-terminal to this residue. It is possible to determine the complete peptide

sequence de novo from its tandem mass spectrum if at least one fragment ion between each adjacent residue pair is present, but a more common approach involves database searching.

Collisionally Activated Dissociation (CAD)

Collisionally activated dissociation, or CAD 50 , (a.k.a. collision-induced

dissociation (CID)), is a very powerful and most commonly utilized tandem mass

spectrometry fragmentation technique. In CAD, a parent ion collides with neutral gas

molecules resulting in subsequent generation of fragment ions. Helium, argon, or

nitrogen gas is typically used as the collision gas.

There are two types of CAD: high- and low-energy CAD. In high-energy CAD,

molecular ions of high kinetic energy (several thousands of electron volts (keVs)) collide

with neutral gas molecules, inducing fragmentation usually upon a single collision. This

fragmentation technique can be utilized on TOF-TOF instruments as well as on sector

instruments. For peptide and protein analysis, high-energy CAD typically produces

cleavage of the peptide bond to generate y and b ions but can also induce fragmentation

of the side chains to produce w and d ions. The w and d ions can provide useful

information such as to differentiate isomeric leucine and isoleucine amino acids; 51

however, they also introduce additional complexity to the mass spectrum and often

complicate its interpretation.

Low-energy CAD is typically utilized in QqQ, ion traps, FTICR, and hybrid

instruments. Low-energy CAD involves molecular parent ions of low kinetic energy

(<1 - 100 eV). These ions, upon many low-energy collisions with gas molecules,

become activated via translation-to-vibration energy transfer. The ion activation is not

caused by electronic excitation but rather by vibrational excitation, and intramolecular

vibrational energy redistribution (IVR) often precedes ion fragmentation. Consequently, low-energy CAD usually results in the dissociation of a bond with the lowest dissociation energy. For peptide and protein analysis, low-energy CAD typically produces y and b ions. Additionally, other labile bonds, such as those formed during protein post- translational modifications (PTMs) (e.g. phosphorylation, glycosylation), often also dissociate during low-energy CAD. This tendency of CAD to produce abundant labile modification losses is one of its main limitations because localization of PTMs is a very important aspect of protein characterizations.

Nonetheless, low-energy CAD is a powerful protein and peptide tandem mass spectrometry technique because it can provide sequence information about the protein/peptide in question. Additionally, low-energy CAD performed on an ESI-triple quadrupole instrument is the preferred technique for identification and accurate quantitation of small biomolecules and biomarkers. This technique has led to a significant, multiplexed expansion of newborn screening for inborn errors of metabolism

(IEM) testing in the early 1990’s, enabling screening for various fatty acid and amino acid metabolic IEM disorders. Finally, positive and negative ESI and APCI QqQ instruments employing low-energy CAD have been recently utilized in various clinical in vitro diagnostics procedures.

Electron capture dissociation and related techniques

Electron capture dissociation (ECD) provides fragments (mostly c and z ions) complementary to those (mostly b and y ions) generated by other tandem MS methods such as CAD. 50 ECD was first introduced by Zubarev et al. in the late 1990s,33 and has since been widely implemented for routine structural analysis of biological molecules. 52

The ECD fragmentation mechanism is believed to be fundamentally different from other

tandem MS methods. It is sometimes referred to as a ‘non-ergodic’ method, 33 viz.

energy gained upon electron capture is used directly to cleave the N - Cα bond prior to

IVR rather than being distributed among various degrees of freedoms within the

molecule to break the weakest bond as in CAD, 50 an interpretation which is the subject

of intense debate. 53-55 ECD is initiated by the capture of low-energy electrons by multiply

charged gas phase molecular ions typically produced by ESI. ECD has been

traditionally performed on an FTICR mass spectrometer because of its ability to confine

low-energy electrons, 31, 36 and its implementation has recently been extended to a radio

frequency ion trap. 56 The capture of the low energy electron (~0.2 eV) is believed to occur in proximity to the protonated site of the molecule, either at a protonated backbone carbonyl site according to the Cornell mechanism, 33, 57 or in the π orbital of an amide group in the presence of a remote charge according to the Utah-Washington mechanism, 58 producing a charge reduced cation radical, with the subsequent backbone

• • N-Cα bond cleavage leading to the formation of c and z fragments. The radical on z

can induce further rearrangements within the molecule, a free radical cascade, 53

producing additional backbone and side chain cleavages both in the proximity of and

remote from the initial radical site. 59 The fragmentation pattern generated by ECD may

vary dramatically depending on the number and location of certain amino acids,

modifications, and charges. 60-64 A number of experimental and computational studies

have been carried out since ECD was first introduced, 33, 65 yet its mechanisms remain

under debate, probably because multiple competing pathways are involved. 54, 66-76

Nevertheless, the capability of ECD to produce unique fragment ions not obtainable by

conventional methods has led to a rapid optimization of ECD and the development of

other ion-electron reaction based tandem MS techniques such as electron transfer

dissociation (ETD), 77 electron ionization dissociation (EID), 78 electron detachment

dissociation (EDD), collectively known as electron activated dissociation (ExD).

ETD was introduced as a substitute for ECD in instruments other than FTICR mass spectrometers, such as a 3D quadrupole or linear ion trap. In ETD, the electron is transferred to the molecular ion from a radical anion to produce fragmentation pattern similar to ECD. Radical anion (the ETD reagent) is produced by a negative CI (nCI) source and the electron transfer reaction occurs in a quadrupole or similar rf-field trapping module. The most commonly used ETD reagents include fluoranthene and azulene.

In EID , ions of interest are irradiated with energetic electrons (>30 eV) resulting

in electron ejection and formation of excited radical ion species, which undergo rapid

dissociation. EID has been successfully applied to analysis of both singly and multiply

charged peptides and proteins. Additionally, EID was implemented in a radiofrequency

linear ion trap instrument along with ECD and other fragmentation techniques for

complementary analysis. 79 The ability of EID to produce ECD-like fragments from singly

charged precoursor ions is unique among all ExD methods, because ECD and ETD are

accompanied by charge reduction upon the electron capture or transfer and cannot be

performed on singly charged ions as the products would be neutral and undetectable.

This feature allows EID to be implemented in instruments with MALDI ionization

sources. 80

ExD has been broadly applied towards the structural analysis of various types of biomolecules, including proteins, oligosaccharides, oligonucleotides, and others. 52, 81-83

Further, ECD and ETD were found to be particularly useful for the characterization of

PTMs in proteins as they can cleave the backbone while preserving the labile groups

and non-covalent interactions upon fragmentation. 84, 85

In-source decay fragmentation

In-source decay (ISD) fragmentation during matrix assisted laser desorption

ionization (MALDI) process is a pseudo tandem MS technique, which allows peptides

and proteins to fragment in a single MS stage. 86 Fragmentation occurs as a result of the

metastable decay of precursor ions during delayed extraction within the ion source. 87 In

ISD, the polypeptide N-Cα bonds are cleaved to generate c and z product ions. These products are similar to those generated in ECD; however, the mechanism of their formation appears to be different. It is proposed that, in ISD, the intermolecular hydrogen abstraction occurs between the peptide and the MALDI matrix producing a hypervalent radical species that undergoes subsequent rapid cleavage of the N-Cα

bond. 88 Therefore, ISD is induced by hydrogen atoms generated by a photochemical

reaction of the matrix and not by electrons as in ECD. 89

The cleavage of the N-Cα bond is normally observed when the following matrices

are used: picolinic acid (PA), 1,5-diaminonaphtalene (1,5-DAN), and 2,5-

dihydroxybenozoic (DHB). 90 These are hydrogen donating molecules with the hydrogen

donating abilities the highest when PA and 1,5-DAN are used together. Different

product ions can be observed in ISD with the use of another class of matrices –

hydrogen accepting matrices. As was recently reported, with the use of such matrices,

e.g. 5-formylsalicylic acid (5-FSA) and 5-nitrosalicylic acid (5-NSA), 91, 92 hydrogen

abstraction from peptides results in the formation of oxidized peptides containing a

radical site at the backbone amide nitrogen with subsequent radical-induced cleavage at

the C α-C bond, leading to the formation of a- and x-type fragments. Some matrices,

such as 2-aminobenzamide (2-AB), 2-aminobenzoic acid (2-AA) have mixed properties, i.e. they can both donate and accept hydrogen atoms generating various types of fragments. 93

The two main limitations of the ISD method are the lack of precursor ion selection for fragmentation and interferences from matrix clusters. It is possible to mitigate these problems by applying HPLC separation prior to MALDI analysis and choosing matrices that produce relatively low amount of cluster ions, and by varying the experimental parameters and instrument setup. Nonetheless, ISD has become a valuable tool for the analysis of peptides and proteins. Importantly, PTMs are preserved during the ISD process. ISD has been applied to analyze peptides and proteins with phosphorylation and O-glycosylation, to differentiate the isobaric amino acid residues leucine and isoleucine, and to obtain information on disulfide-linkages. 94-96 Alternatively, disulfide bonds can be reduced allowing the analysis of Cysteine-containing peptides. 97 Further, it was shown that only limited hydrogen scrambling is observed during the ISD fragmentation process. 98 This is beneficial for the hydrogen/deuterium exchange studies of proteins to obtain information on their dynamic and structural properties. Finally, ISD has been implemented in MS imaging of tissue samples to simultaneously identify and localize proteins. 99

1.2.4 ESI QqQ-FTICR mass spectrometer

One of the instruments used for the work described in this thesis is a custom-built hybrid electrospray triple quadrupole FTICR mass spectrometer (ESI qQq-FTICR

MS). 100 The instrument scheme is demonstrated in Figure 1.8.

Figure 1.8 The ESI qQq-FTICR mass spectrometer. Repr oduced (modified) from

O'Connor, P. B. et al ., Rapid Communications in Mass Spectrometry, 20 , 259-266,

2006 , with permission.

In Figure 1.8, the direction of ions traveling through the instrument is from right to left. The ion source is a direct infusion nanoESI source. The in-house pulled glass capillary tips are used to generate a ~50 nmol/min flow and the nanoelectrospray by applying 1100-1300 V to a metal wire inserted into the tip. Ions are sucked into the vacuum interface region of the instrument at ~10 -3 Torr by a combination of the electric

field and the pressure gradient. Ion desolvation is accommodated by the curtain gas

applied between the orifice and the curtain plate. Generated ions are focused by an RF-

only quadrupole Q0, precursor ions are isolated in a mass analyzing quadrupole Q1, and

accumulated and/or fragmented in a collision cell Q2 (modified from an AB/MDS Sciex

API 365 instrument). IQ1, IQ2, and IQ3 are focusing lenses, and typical voltages

applied to these lenses as well as the orifice (OR), ring electrode (RNG), and skimmer

(SK) are denoted. A potential gradient is created by applying decreasing voltages

across the instrument from the front to the end to efficiently transmit the ions and to

decrease their initial kinetic energy. Stubbies (ST) is a set of short quadrupoles used to

smoothen transition of ions from Q0 to Q1. Linac is a set of four rods used to create an

axial electric field to push ions into a tighter packet towards IQ3 before ion extraction to

minimize the transfer efficiency reducing time-of-flight effect. After ion accumulation, the

IQ3 is pulsed to a negative value to allow ion extraction into the next module – hexapole

1 (before the gate valve) and sectioned hexapole 2 (after the gate valve). These hexapoles are used for ion transfer to the ICR cell. Sectioned hexapoles allow for differential pumping with the use of conductance limits to maintain the UHV (~10 -10 Torr) in the ICR cell region. The gate valve separates the instrument into two sections. When closed, it allows the front end of the instrument to be opened up for cleaning and other purposes without disturbing the UHV in the ICR cell region. The ICR cell is a capacitively coupled closed cylindrical cell with two end trapping plates positioned inside the homogeneous region of a 7 T magnetic field produced by an actively shielded magnet (Cryomagnetic Inc., Oak Ridge, TN). After ions enter the ICR cell, the front

(hexapole) and back (filament) trapping plate voltages, V qtrap and V ftrap , respectively, are ramped to high positive value (~15 V) to trap the ions inside the cell. The trapping plate voltages are reduced to ~1 V before ion excitation to minimize the influence of ion magnetron motion. The inner trapping plates are also used to confine the ions in the homogeneous part of the magnetic field along the axis of the instrument by applying a small voltage of ~1 V (V inner - rings ). All voltages above are listed for the analysis of positive ions, the polarity is inverted for the analysis of negative ions. The center plates are used for ion excitation and detection.

101 An indirectly heated dispenser cathode (E gun , Heatwave, CA, USA) and a grid

(Grid) are used for ECD experiments. The cathode has a BaO surface which, when heated by passing a current (1.5-1.8 A) through a resistor, emits electrons. A pulsing negative voltage is applied to eject the electrons from the cathode surface, typically at -

0.2 V to -1.5 V for generation of low-energy electrons. The grid is used to extract

electrons from the cathode for efficient transfer to the ICR cell by applying a constant positive voltage of ~10 V. The time period during which electrons are emitted from the cathode is called the electron irradiation time and is usually within a 5-100 ms range. At all other times, the cathode potential is set to 9.9 V to avoid introducing electrons into the

ICR cell. The combination of magnetic and electric fields of an FTICR allows for simultaneous radial confinement of ions and electrons inside the cell and thus their reaction. Upon the electron beam passing through the center of the cell and the ions rotating in the center of the cell, the electron capture occurs to generate fragments as was explained previously. By varying the cathode potential it is possible to eject electrons of different energies for performing alternative ECD-like fragmentations, such as hotECD and EID. In addition, other fragmentation techniques can be performed on this instrument. These include CAD - either performed in the collision cell (Q-CAD), or inside the ICR cell (SORI-CAD), 34 and IRMPD. 35 This instrument was extensively used for the analysis of peptides, proteins, and their PTMs, as well as carbohydrates, 102 typically providing high mass resolving power of >100,000 and ~2 ppm mass accuracy with internal calibration.

1.2.5 Bruker Daltonics solariX FTICR MS

Another instrument used for the work presented in this thesis is a commercial hybrid Qh-FTICR mass spectrometer with a 12 T actively shielded magnet, named solariX TM (Bruker Daltonics, Billerica, MA, USA). The instrument schematic is shown in

Figure 1.9 (Bruker Daltonics) with the exception that only an ESI ionization source was available in the current configuration.

Figure 1.9 The solariX TM FTICR mass spectrometer (Bruker Daltonics).

The atmospheric pressure nanoESI is implemented using either an in-house pulled glass capillary tips or a robotic Nanomate source (Advion, Ithaca, NY, USA).

Ions, transmitted through a heated glass capillary positioned perpendicular to the axis of the instrument, are further guided by the electrostatic and RF multipole ion optics to the

Quadrupole for isolation. Isolated precursor ions are then transferred to the Collision

Cell (hexapole), which is used for ion fragmentation using either CAD or ETD, as well as for ion accumulation. The anion radical reagent is introduced to the collision cell from an external negative chemical ionization (nCI) source via a split octapole to induce ETD.

Unfragmented precursor and fragment ions are transferred to the ICR cell via a Transfer

Hexapole. The ICR cell is a closed cylindrical Penning trap. A hollow indirectly heated dispenser cathode is used to provide electrons for ExD experiments. A 25 W continuous wave (CW) CO 2 laser (10.6 µm, Synrad, Mukilteo, WA) is used for IRMPD experiments.

The hollow cathode 103 allows for simultaneous irradiation of the ion cloud by electron and

photon beams to perform activated-ion ECD (AI-ECD). In addition, SORI-CAD can be

performed inside the ICR cell. This instrument can be connected to an HPLC module for

high throughput HPLC-FTICR MS proteomic analysis with a variety of fragmentation

techniques available for protein identification and characterization of PTMs. The typical

resolving power is ~200,000 at m/z 400, and the typical mass accuracy is <0.5 ppm with internal calibration, and <2 ppm with external calibration.

1.3 High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is an analytical technique for analyte separation. An HPLC instrument contains four major components: a stationary phase packed into a column, HPLC pump(s), a mobile phase, and a detector. The separation of analytes in a mixture is based on the difference in interaction of analytes with the stationary phase and the mobile phase. Depending on the stationary phase and the mobile phase used, there are many different types of HPLC. The ones that are used for biomolecular sample preparation and sample separation are the size exclusion, ion exchange (either anion or cation), normal phase HPLC (NP-HPLC), and reversed phase

HPLC (RP-HPLC).

Size exclusion chromatography (SEC), as its name implies, separates analytes based on their size. The basis for this technique is the physical, rather than chemical interaction of the analyte molecule with the stationary phase. As analytes move through the SEC stationary phase, smaller analytes can be temporarily trapped within the

"pores" of the stationary phase whereas larger analytes cannot enter these pores and pass straight through; therefore larger analytes elute faster than smaller analytes. SEC

is often used for purification of large biological molecules such as proteins, DNA/RNA

and carbohydrates.

Ion-exchange chromatography separation is based on the electrochemical interaction between the ions or the polar analytes with the stationary phase. The stationary phase for ion-exchange chromatography has ionic functional groups that interact with the oppositely charged analyte ions, thus providing the separation – the stronger the interaction, the longer the retention time. Counter ions are often added to the mobile phase (a.k.a. pH modifier) to affect the strength of interaction and the retention times. Ion-exchange chromatography is often used to separate and purify proteins and peptides, as well some classes of carbohydrates, such as glycosaminoglycans (GAGs).

Normal-phase HPLC separates analytes based on their interactions and adsorption to a polar stationary phase. NP-HPLC employs non-polar mobile phase and works well for analytes that can be dissolved in non-polar solvents. The more polar analytes interact more strongly with the polar stationary phase, which in turn results in longer retention time. The stereochemistry of the analytes also plays a role in retention and separation in NP-HPLC. The analyte retention and elution can be manipulated by increasing or decreasing the polarity of the mobile phase; however, when the mobile phase becomes too polar or aqueous, it will interact with the stationary phase and subsequently deactivate it. The NP-HPLC has not been used widely for biomolecular separation and analysis as most of the biomolecules are not soluble in highly non-polar solvents, and it eventually gave way to the reversed-phased HPLC for biomolecular separation and analysis (Section 1.3.1). However, recently a variation of NP-HPLC,

known as the hydrophilic interaction liquid chromatography (HILIC), has been used for

biomolecular applications. 104

HILIC stationary phase is polar and often based on such functional groups as

amines, amides and zwitterionic groups. The HILIC mobile phase is non-polar in nature

and often a mixture of acetonitrile and water. In HILIC, analyte retention and separation

are based on two factors: the electrochemical interaction and liquid-liquid

extraction/retention. The electrochemical interaction between the analytes and the

HILIC stationary phase is a weak (e.g. hydrogen bonding) interaction facilitated by

employing a polar mobile phase. The liquid-liquid extraction and retention is caused by

formation of a water-rich layer at the surface of the stationary phase (without

deactivating it as in the traditional NP-HPLC), and subsequently the analytes are

dispensed between this layer and the mobile phase based on their hydrophilicity. The

ability to utilize water as well as volatile organic solvent such as acetonitrile makes

HILIC-HPLC a very attractive separation method for electrospray ionization mass

spectrometry applications. HILIC-HPLC is often used for HPLC-ESI-MS (MS/MS)

analysis of proteins, peptides and other biomolecules. 105

1.3.1 Reversed Phase High Performance Liquid Chromatography (RP-HPLC)

Reversed-phase HPLC (RP-HPLC) was introduced as an analytical separation technique in the mid 1970’s. 106 The name “reversed” comes from the fact that RP-HPLC

adopts an inverted philosophy compared to the normal phase HPLC: whereas NP-HPLC

utilizes polar stationary phase and non-polar mobile phase, RP-HPLC employs non-

polar stationary phase and polar mobile phase. Since its introduction, the RP-HPLC has

been used so extensively (including in biomolecular analysis), that often RP-HPLC and

RP-HPLC-MS applications are no longer denoted as reversed-phase and are assumed

to be.

The most often used stationary phase in RP-HPLC is silica with bonded straight chain alkyl groups. 106 In RP-HPLC, samples are loaded onto the HPLC column using

relatively polar solvents (i.e. with a higher water content). The analytes are retained on

the column by hydrophobic interactions with the non-polar stationary phase. These

hydrophobic non-covalent interactions occur between the hydrophobic parts of the

molecules and the alkyl chains of the stationary phase; therefore, analytes with a larger

hydrophobic patch interact more strongly with the stationary phase, and are retained on

the column for a longer period of time. The analytes are eluted off the column by

ramping up the organic (non-polar) content of the mobile phase. The basic process of

elution is a result of the analyte/mobile phase hydrophobic interaction overcoming that

between the analyte and the stationary phase. There are a number of factors affecting

analyte retention and elution: stationary phase chemistry, stationary phase particle and

pore size, column size and temperature, mobile phase composition and mobile phase

gradient, mobile phase pH modifier (a.k.a. solvent additive) and the flow rate.

Silica with bonded straight alkyl chain of four, eight and eighteen carbons are the most commonly used RP-HPLC stationary phases, which are commonly referred to as

C4, C8 and C18, respectively. These stationary phases interact differently with different analytes. C4 is most often used for protein separation and analysis. The reason for using a shorter alkyl chain for protein separation is that proteins contain large hydrophobic regions, which may interact with stationary phases employing longer alkyl chains too strongly to be efficiently eluted. On the other hand, C18 is often used for small molecule and peptide analysis. Unlike proteins, small molecules and peptides

have smaller hydrophobic molecular areas, thus necessitating longer alkyl chain

stationary phase for better separation. C8 stationary phase is often considered as a

universal RP-HPLC stationary phase and works well for peptide and small molecules

separation and analysis.

Analyte retention and separation are also affected by the RP-HPLC column sizes

(inner diameter (i.d.) and length). For LC-MS applications, relatively small analytical, and not larger preparative, HPLC columns are typically utilized. Typical RP-HPLC columns used for LC-MS analysis of biomolecules have either 2.1 mm or 4.6 mm i.d. and are 10 to 250 mm in length. Generally, as the column volume increases, the analyte retention time increases as well.

Additionally, column’s stationary phase particle size and pore size play a role in analyte separation and retention. Typical RP-HPLC columns are packed with stationary phase beads or particles. These particles are spherical and porous in nature. The diameter of the particles is known as the particle size and the size of the pores is known as the pore size. The particle and pore sizes play off each other to determine the analyte retention and separation. Smaller particle size or pore size or both mean higher surface area of the stationary phase, thus more extensive interaction with analytes leading to longer retention. This also requires higher mobile phase column pressure.

Typical LC-MS analysis of biomolecules utilizes RP-HPLC columns with particle size less than 10 microns and pore size between 100 and 300 angstroms ( Ǻ).

Both the column temperature and the mobile phase flow rate affect the HPLC performance. Because analytes interact with the stationary phase via hydrophobic interactions, changes in temperature will change the interaction strength, which in turn will affect analyte retention and separation. It is also desirable to control the column

temperature to achieve reproducibility and consistency in column performance and analyte separation. Flow rate also has an effect on analyte retention: generally higher flow rate leads to faster elution off the column, but this relationship is not linear.

Mobile phase composition and mobile phase gradient have a pronounced effect on analyte separation and elution in RP-HPLC. Typically, a sample is loaded onto the

RP-HPLC column using an organic/water mixture which is relatively polar, i.e. high in water content. The organic solvent commonly used for LC-MS biomolecular analysis is either methanol or acetonitrile. Not only are these organic solvents optimal for reversed- phase chromatography, but they are also ideal for ESI and APCI ionization sources owing to their high evaporation efficiency. As the mobile phase is ramped up to higher organic/lower water content and becomes more non-polar, the hydrophobic interactions between the analytes and the stationary phase decrease resulting in eventual analyte elution. The organic solvent used and the water content, as well as the steepness of the gradient ( % change in water content over time) all influence the analytical performance of RP-HPLC.

Another important factor determining the analyte retention and elution is the pH modifier. The pH modifiers are typically inorganic salts or weak acids that are added to water, or organic solvent, or both. The pH modifier increases the surface tension of the mobile phase which in turn tends to increase analyte retention and improve the separation. Some of the best pH modifiers that are often used for LC-MS applications are acetic acid (AA), formic acid (FA), triflouroacetic acid (TFA) and heptafluorobutyric acid (HFBA). These solvent additives not only improve the chromatographic separation and resolution, but also act as proton donors for analyte ionization. It is worth noting that

TFA tends to suppress ESI and APCI generated ions and should only be used at very low concentrations (0.01 – 0.05 %). 107

1.3.2 On-line and off-line Liquid Chromatography Mass Spectrometry

Coupling HPLC to mass spectrometry results in hybrid analytical technique known as liquid chromatography-mass spectrometry (LC-MS). The ability of liquid chromatography to separate analytes in time and space provides additional specificity and selectivity to the mass analysis. Additionally, coupling LC to mass spectrometry analysis often improves sensitivity by sample complexity reduction, analyte concentration on column, and/or removal of impurities from the analytes of interest. In on-line LC-MS, the LC effluent off the column is directly connected to a mass spectrometer and the data acquisition happens in real-time. ESI, APCI, and atmospheric pressure photoionization (APPI) ionization sources are very amendable to on-line LC-MS, and specifically RP-HPLC-MS (or MS/MS). The reasons are appropriate flow rates coming off analytical HPLC column (~10 – 500 µl/min for ESI, and ~300 – 900

µl/min for APCI and APPI) and appropriate HPLC solvents (high organic content acetonitrile:water or methanol:water mixture). HPLC-ESI-quadrupole based LC-MS (or

MS/MS) systems are widely used for biomolecular analysis due to the quadrupole scanning nature. For on-line HPLC-ESI-TOF systems, because of the pulsed nature of a TOF mass analyzer, ions need to be trapped (usually in a front end quadrupole), and then pulsed into the TOF analyzer, as employed in Q-o-TOF instruments. This happens on the millisecond scale and the HPLC performance or resolution is not affected.

In off-line LC-MS experiments, the LC effluent is not directed introduced into the mass spectrometer in real-time. In this case, the LC effluent is typically collected in

different fractions (as a function of time), which are later mass analyzed as a liquid (in

ESI-MS) or deposited on the MALDI plate. There are a number of reasons to perform off-line LC-MS analysis. Primarily, some ion sources, especially those that operate under vacuum (e.g. EI, CI, and MALDI) are not amendable to LC-MS. Another reason to perform off-line LC-MS analysis is the incompatibility of LC effluent with liquid based ionization sources. For instance, the majority of normal-phase HPLC techniques utilize

ESI or APCI unfriendly mobile phase (e.g. hexane). Therefore, in this case, the LC effluent needs to be collected as fractions and solvent exchanged to ESI/APCI amendable solvent type. Off-line HPLC MALDI-TOF technique is an extremely powerful technique for protein analysis, where a protein trypsin digest is subjected to HPLC separation and off-line fractionation, which are subsequently deposited onto a MALDI plate for MALDI-TOF analysis.

1.4 Introduction to deamidation and isomerization of peptides and proteins

1.4.1 Deamidation and isomerization of peptides and proteins

Deamidation of asparagine (Asn) and isomerization of aspartic acid (Asp) residues, are two of the most common PTMs found in all proteins. Under physiological conditions, deamidation of Asn proceeds through formation of a cyclic succinimide intermediate by the nucleophilic attack of the backbone nitrogen atom in the following peptide bond on the carbonyl group of the Asn side chain (Figure 1.10). Upon hydrolysis of the succinimide intermediate, two isomeric products, Asp and isoAsp are formed, typically in a 1:3 ratio in short unstructured peptides. 108, 109 In proteins, this ratio could be different from 1:3 and is influenced by higher-order structures. 110, 111 Asp can also be

directly isomerized into isoAsp through the same cyclic intermediate due to the dehydration process. 112 The succinimide formation from Asp is ~ 10 - 40 times slower than from Asn at neutral pH, yet the reaction rate may vary greatly depending on the adjacent residues, protein conformation, the proximity of the Asp/Asn residue to the protein surface and the molecular environment. 108, 112-114 For example, the first-order deamidation half-time of an Asn residue surrounded by Gly residues (at pH 7.4, 37.0 ◦C, in 0.15 M Tris HCI) is 1 day; however, if the C-terminal residue is Pro, it would be

≥ 32 years. 115 It seems that the residue that follows the Asn residue is the most important factor on its deamidation rate, and the fastest times are for the following residues: Gly>His>Ser>Ala, etc. 115

Figure 1.10 Isomerization of aspartic acid and deamidation of asparagine via a succinimide intermediate.

Under physiological conditions (at pH ≥7), both Asp isomerization and Asn deamidation are spontaneous nonenzymatic reactions, which proceed via a 5- membered ring succinimide intermediate. 108 At pH (4-7), deamidation proceeds by the initial formation of a tetrahedral intermediate that can be converted to succinimide.

Deamidation rates reach the minimum at pH between 3 and 4. At low pH (<3), Asn deamidation occurs by direct acid-catalyzed hydrolysis to form the only product aspartate. 116 In addition, an increase in temperature leads to an increase in the reactivity of Asn and Asp residues in proteins leading to faster deamidation and isomerization rates. 117 These factors often lead to artifactual deamidation and isomerization during sample preparation and handling, thus, have to be considered when the protein characterization and PTMs analysis are performed.

1.4.2 Biological importance of deamidation and isomerization

Asn deamidation and Asp isomerization are common in vivo . They are particularly common in long-lived proteins and have been proposed as molecular timers of biological events. 112 These modifications are often associated with aging, 114 eye lens abnormalities, 118 amyloid diseases such as Alzheimer disease 119, 120 and Type 2 diabetes mellitus, 121 and apoptosis. 122

Structural change. By introducing an additional methylene group into the polypeptide backbone (isoAsp) and a negative charge to the polypeptide (deamidation), these modifications may alter protein conformation, activity and stability, and trigger protein aggregation. 114, 119, 123 For example, the 3-dimensional crystal structure of

Ribonuclease U2B revealed that formation of isoAsp32 led to a single turn unfolding of the α-helix to form a U-shape loop structure, affecting the hydrolytic activity of the

protein. 124 Single Asp isomerization was shown to deactivate the antigen-binding region of the immunoglobulin gamma (IgG)-2 antibody. 125 The presence of the isomerized Asp may also affect the proteolytic stability of the protein. For example, Lys-C proteolysis is hindered when the Lys residue is adjacent to an isoAsp in IgG-1 antibody; 126 isoAsp residues are also resistant to Asp-N proteolysis. 127-129 On the other hand, carboxypeptidase Y cleaves N-terminally to an isoAsp residue recognizing its α- carboxylic acid as if it is a carboxyl-terminal amino acid. 130

Protein L-isoaspartyl O-methyltransferase . Although in vivo Asn deamidation is an irreversible reaction, the isoAsp accumulation can be minimized by protein L- isoaspartyl O-methyltransferase (PIMT), which catalyses isoAsp conversion to Asp

(registry number: EC 2.1.1.77). 120, 131, 132 PIMT catalyzes the transfer of a methyl group from S-adenosy-L-methionine to the α-carboxyl group of L-isoAsp. As a result of the spontaneous hydrolysis of the methyl ester, the succinimide intermediate is formed, which again, upon hydrolysis, would generate a mixture of Asp and isoAsp. Multiple actions of PIMT enzyme can minimize isoAsp content with time. PIMT is highly conserved, ubiquitously expressed in many tissues, mostly as a cytosolic enzyme. Loss of PIMT has damaging consequences, especially in neuron cells. For example, in PIMT deficient mice, isoAsp accumulated in the cytosolic fraction of brain, heart, liver, and erythrocytes. The knockout mice showed significant growth retardation and fatal seizures at ~6 weeks after birth, suggesting that PIMT is essential for normal growth and central nervous system function. 133 Overall, formation of isoAsp residues and their subsequent correction by PIMT are widely believed to constitute an important pathway of protein damage and repair. 120, 132

Potential biomarkers . The study of deamidation and isomerization processes has significant consequences for the identification of proteins and the search for potential biomarkers. For example, among several promising biomarkers for the risk of developing retinopathy of prematurity (ROP) disease, deamidation of globin chains may indicate underlying prenatal pathologic mechanisms in ROP developing. 134 In another example, the asparagine deamidation in peptides containing the Arg-Gly-Asn (RGN) sequence generates Arg-Gly-isoAsp (RGD) motif that is being exploited for ligand- directed targeted delivery of various drugs to angiogenic vessels and as a potential biomarker of related pathology. 135

Pharmaceutical products. Finally, In vitro Asp isomerization and Asn

deamidation can also occur during protein production and storage. IsoAsp buildup could

be detrimental to the protein structural integrity and stability, which affects the shelf-life

and potency of the therapeutic monoclonal antibodies (mAb) and other protein-based

new drug entities in the pharmaceutical industry. 136 For example, spontaneous

deamidation of Protective Antigen protein (PA), a key component in an anthrax vaccine,

leads to its inactivation. 137

1.5 Introduction to β-peptides

β-Amino acids . Similar to the isoAsp, a β-amino acid has an extra methylene group incorporated between its amino and carboxylate groups. There are two types of

β-amino acids: β2 and β3, with the side chain attached to α and β carbon respectively

(Figure 1.11). β-Amino acids do not normally occur in nature except for β-alanine (a component of pantothenic acid, of coenzyme A, and of carnosine in muscle tissue) and

β-aspartate (isoAsp); neither do β-peptides, but those can be synthesized. 138, 139 It

should be noted, that naturally occurring β-alanine, and β-aspartic acid have the same total number of carbons as their α-analogues – one more in the backbone and one fewer on the side chain; however, β-amino acids normally used for β-peptide synthesis often

have an extra carbon within the backbone, but contain the same side chain as those in

α-amino acids, and thus are called β-homo-amino acids (in this thesis, all β-amino acids

in synthetic peptides are β-homo-amino acids).

Figure 1.11 β-Peptide nomenclature.

β-Peptides . β-Peptides were first synthesized and examined in the mid-1990s

by Seebach. 138 Since then, they have been intensively studied on their structural and

biological properties, and on their interactions during peptide folding. Because the

backbone of β-peptides is longer than that of α-peptides, they form different secondary

structures, which appear to be more stable and have richer conformational energy

surface compared with their α-analogues. 138, 140 They also fold into helices or hairpin-

type structures, but with larger variety than α-peptide secondary structures. In contrast

to α-peptides, β-peptides form such structures with shorter chain lengths starting at 4 residues. Furthermore, there are differences in similar structures, such as screw sense of the helix and the macrodipole direction; as opposed to α-peptidic parallel pleated

sheet, in that of β-peptides, all C=O bonds point in the same direction and all N-H bonds

in the opposite one. 140

Biological significance . The biologically important property of β-peptides is their

stability against proteolytic degradation and other enzymes in human and various living

organisms. 141-143 However, β-peptides can be designed in a way that they would

resemble the epitopes of corresponding natural peptides to mimic their function and be

used as agonists and antagonists in peptide-protein, protein-protein, and peptide-

DNA/RNA interactions 144-146 . These features provide great potential for β-peptides in biomedical application as proteolytically stable therapeutics and antibiotics for antibiotic- resistant pathogens.

There are a number of examples of β-peptide application in mimicking the natural

α-peptidic properties. A helical β3-peptide was designed that recognizes a cleft on the

surface of the human oncogene product double minute 2 (hDM2) mimicking an α-helix of

the major tumor suppressor p53 protein in its activation domain. 145 When bound hDM2

inhibits p53, however, binding of the β3-peptide to hDM2 inhibits the hDM2 and p53

interaction allowing p53 to proceed its function, which is important for cancer therapy.

Another β-peptide with an amphiphilic helix was developed to mimic natural antibiotic of

the magainin’s class and was shown to be effective against bacterial species that are

resistant to common antibiotics - the bacterium Enterococcus faecium A436

(vancomycin resistant) and Staphylococcus aureus 5332 (methicillin resistant). This

peptide does not induce haemolysis (the erythrocyte rupture), which is essential for

successful therapeutic application. 147

Methods of analysis . Circular dichroism spectroscopy, X-ray crystal-structure

analysis and nuclear magnetic resonance spectroscopy are primarily used to study the

structure of β-peptides. 138, 140, 142, 144 Radioactive labeling, HPLC and MALDI mass spectrometry have also been applied to monitor β-peptides in tissues. 141, 143 However,

an improved, fast and accurate analytical method capable of distinguishing α from β, and

β2 from β3 type amino acids would be of a great utility for structural confirmation and

analysis of β-amino acid-containing peptides.

1.6 Thesis overview

This thesis focuses on the study of peptide and protein deamidation and

isomerization by electron capture dissociation tandem mass spectrometry and liquid

chromatography. IsoAsp is a β-type amino acid, thus other types of β-amino acids are also studied by ECD as well as ISD tandem MS. The deamidation and isomerization research includes: Chapter 2, isoAsp analysis in amyloid β fragments; Chapter 3, the analysis of isoAsp, Asp, and Asn containing peptide variants by LC; Chapter 4, the differentiation of the N-terminal Asp and isoAsp in peptides; Chapter 5, isoAsp in amyloid β fragments in CSF. β-Peptide studies include: Chapter 6, fragmentation of β- linked peptides by tandem MS using ECD and ETD; Chapter 7, in-source decay for β- peptide analysis. Chapter 8 is conclusion and future work. Overall in this thesis, new and improved methods are suggested for characterization and differentiation of β-amino acids.

Chapter 2

Identification of isoAsp in Amyloid βββ Peptides by Tandem ExD MS/MS

2.1 Introduction

2.1.1 Amyloid β in Alzheimer’s disease.

Amyloid beta (A β) peptides are the major components of the vascular and plaque

amyloid filaments in individuals with Alzheimer's disease (AD). Various forms of A β are proteolytically cleaved from the A β precursor protein, with A β1-40 and A β1-42 being the most abundant forms found in Amyloid deposits. 148 Ever since A β was first purified and

characterized, it has been strongly associated with the pathology of AD, 149, 150 although it

remains unclear what initiates the disease. According to the most widely accepted

hypothesis, cerebral A β accumulation is the primary cause in AD. The rest of the disease process stems from imbalance between A β production and clearance. 151 Many

attempts have been made to measure the concentration of A β peptides in biological

fluids, but it is difficult to correlate A β levels with disease stage and, thus, to use it as an

AD biomarker. 152 Further research and development of analytical methods is necessary

for early AD diagnosis, disease progression, monitoring, and better understanding of the

disease.

______1This chapter has been partially reproduced with permission from Sargaeva, N. P.; Lin, C.; O'Connor, P. B., Identification of aspartic and isoaspartic acid residues in amyloid beta peptides, Including A beta 1-42, using electron-ion reactions, Analytical Chemistry , 2009 , 81 , 9778-9786. Copyright 2009, American Chemical Society. 57

2.1.2 Isoaspartic acid formation

Isomerization of aspartic acid (Asp) is one of the most common post translational modifications (PTMs) in all proteins that accumulate with age in long-lived proteins, especially in tooth, bone, cartilage, lens, and brain tissues. 114 The isomerization product

is isoaspartic acid (isoAsp). It can also be formed from asparagine deamidation (Figure

1.10). At physiological pH both reactions proceed via an entropy driven 153 formation of

the five-membered succinimide ring intermediate followed by a rapid hydrolysis. As a

result, aspartic and isoaspartic acid residues are typically formed in a ratio of 1:3. 108, 109

Moreover, at pH ~7.4, IsoAsp formation is preferential because of the higher acidity of

the isoAsp side chain. 153 Under physiological conditions, both Asp isomerization and

Asn deamidation are spontaneous nonenzymatic reactions. 108, 109, 114, 120, 153, 154 The reaction rates depend mainly on the nature of the adjacent residues, the higher order structure of the protein, and the molecular environment. 108, 112, 114 Formation of isoAsp is

speculated to change the protein structure as it introduces an additional methylene

group into the polypeptide backbone. This can change protein function and activity, or

trigger aggregation. 114, 119, 123 In addition, proteins containing isoAsp may not fully degrade as isoaspartate residue hinders proteolytic degradation.155 Nonetheless,

harmful isoAsp products can be partially repaired by the intracellular enzyme, protein

isoaspartyl methyltransferase (PIMT), which selectively converts isoAsp residues back to

the succinimide intermediate. 114, 120

2.1.3 IsoAsp in Alzheimer’s disease

Isomerization of aspartic acid is directly related to the pathology of Alzheimer’s

disease. A β peptides have 3 aspartic acids in the sequence at the 1 st , 7 th , and 23 d

residues and formation of isoaspartate is enhanced in A β peptides in AD. Roher et al. found that Asp1 and Asp7 were isomerized in the cerebral plaque samples of Alzheimer patients. 119 Recently, isoAsp7 and isoAsp23 were found in the core of senile plaques and Amyloid-bearing vessels as was shown with anti-isoasp7 and anti-isoasp23 antibodies. 120 Moreover, the Iowa (Asn23) 156 and Japanese-Tottori mutations (Asn7) 157

in familial AD have a potential to accelerate formation of isoAsp, presumably due to

asparagine deamidation. Accordingly, isoAsp23-containing A β peptides were preferentially detected in vascular deposits in Iowa cerebral Amyloid angiopathy brain. 158

It was further suggested that spontaneous isomerization at position 23 induces the

conformational change to form a β-turn of the polypeptide chain. This, in turn, plays a pathogenic role in the deposition of A β peptides in sporadic AD. 120 In vitro experiments

showed increased fibrillogenesis and enhanced neurotoxicity of isoAsp23-containing A β peptides, 120, 159 however, other results suggested that A β aggregative ability and

neurotoxicity were not enhanced by this modification. 160 Similar studies of A β with

isoAsp7 revealed that it is primarily deposited in the core of senile plaques. IsoAsp7

accumulation was associated with the age of the plaque as the number of isoAsp

positive plaques increased in parallel with the disease severity. 161

There have been many studies of isomerization events in A β peptides, but its

role in AD pathogenesis is still unclear. Formation of isoAsp in A β might be a reason for

its impaired degradation leading to accumulation. This aggregation could be a way for

biological systems to reduce the toxicity of the non-degradable A β peptides. Further

research is needed to better understand the role of isoAsp formation.

2.1.4 IsoAsp detection methods

The detection of isomerization products is analytically challenging due to their

similar chemical properties and identical molecular mass. Nevertheless, various

methods have been applied to differentiate and quantify the isomers, including

immunological methods based on detection by specific isoAsp antibodies, 120, 158, 161 liquid

chromatography, 119, 154 PIMT enzyme utilizing assays, 119, 154 and tandem mass

spectrometry (MS) analysis. 162-164 New methods addressing isoAsp detection are quickly

emerging in the field to better understand the harmful effects isoAsp accumulation can

generate in biological systems and pharmaceutical applications. These new methods

often utilize a combination of existing methods, such as high-performance liquid

chromatography (HPLC) separation followed by MS analysis. 165, 166 In addition,

immunochemistry analysis, capillary electrophoresis, HPLC-MS, or tandem MS alone,

can be applied to analyze l-Asp, l-isoAsp, d-Asp, and d-isoAsp isomerized/racemized peptides. 167-169 Upgraded HPLC, known as the ultra performance liquid chromatography

(UPLC) system, could further separate isoAsp and Asp located directly at the N-

terminus. 170

Edman degradation is a chemical cleavage method useful for IsoAsp identification because it does not cleave the isopeptide bond. Similarly, enzymatic digestion with the endoproteinase Asp-N does not cleave N-terminal to the isoAsp acid.110, 127-129, 171, 172 This was utilized in combination with 15 N isotope labeling and MS for the detection of IsoAsp. 127 The 18 O labeling coupled to MS can also be used for

detection of Asn deamidation and Asp isomerization;173, 174 however, this can only be

applied to detection of in vitro modification sites in the protein, but not to identify

modifications already existing in biological samples prior the analysis. In addition, Alfaro

et. al. recently introduced a new method for the affinity enrichment of isoaspartyl proteins, where chemo-enzymatic detection using the PIMT enzyme and hydrazine trapping were applied. 175

Many of the methods for studying deamidation utilize mass spectrometry, which over several decades, has become one of the most powerful tools in biological sciences.

Although a single stage of MS cannot normally distinguish isomers, because they have identical masses and are represented as a single peak in the mass spectrum, tandem

MS has shown some successful results. For example, low-energy collisionally activated dissociation (CAD) has been applied for Asp/isoAsp differentiation based on the difference in the abundance of the immonium and b and y ions, 163, 164 as well as the

162 b + H2O and y - 46 ions that resulted from cleavages N-terminal to an isoAsp residue.

However, the abundance of all above-mentioned ions can be strongly influenced by the peptide sequence, and in some cases, diagnostic ions were not observed. Therefore, these methods require comparative analysis with standard peptides, which is not always possible for the analysis of biological systems.

An alternative tandem MS-based method was recently developed, where isoaspartic residues in peptides 176 and proteins 177 were unambiguously identified and quantified 178 by Electron Capture Dissociation (ECD). In ECD, electron capture results

• 33 in the N-Cα bond cleavage producing c and z type fragments (Figure 2.1.a).

Furthermore, the radical created by electron capture can initiate additional backbone

53, 179 and/or side chain cleavages. ECD can also result in the cleavage of Cα-Cβ, bond and formation of specific fragments in Asp- and isoAsp-containing peptides. For Asp residues, this leads to the loss of a CH 2C(OH) 2 molecule from the charge-reduced species and the formation of the [M + nH - 60] (n-1)+• ion; for isoAsp residues, cleavage of

the Cα-Cβ bond will result in a breakage of the polypeptide chain and formation of the

• cm + 57 and z n-m - 57 fragment ions will be generated (Figure 2.1.b).

2.2 Experimental section

2.2.1 Peptides and reagents

Standard A β1-40 was purchased from Sigma Aldrich (St. Louis, MO, USA). The isomerized form of A β17-28 (LVFFAEisoDVGSNK, custom synthesized), and A β1-42 with Tottori - Japanese Mutation at [Asn7] were obtained from AnaSpec (San Jose, CA,

USA). Sequencing grade trypsin was purchased from Roche Diagnostics (Indianapolis,

IN, USA).

2.2.2 Sample preparation

Aβ1-40 was digested with trypsin in 100mM Ammonium Bicarbonate buffer at an enzyme : substrate weight ratio of 1:50 in µg/µl concentration, overnight at 37°C with prior incubation at 65°C for 20 min. A β1-42 [Asn7] was deamidated overnight at 37°C in

0.4 % aqueous Ammonium Hydroxide with pH >10.2 in µg/µl concentration. After digestion or deamidation, samples were dried in a SpeedVac system to stop the reaction and evaporate volatiles.

Figure 2.1 The ECD major pathway to produce c and z fragment ions (a); proposed ECD mechanism for formation of diagnostic fragment ions (b).

2.2.3 Mass Spectrometry

Mass spectra were acquired on a custom built qQq-FTICR MS with a nanospray

source and a 7 T actively shielded magnet. 100, 102 Samples were electrosprayed at 1-

5 µM concentration in 50:50 MeOH:H 2O with 1 % formic acid. Ions were isolated in the

first quadrupole Q 1, accumulated in the second quadrupole Q 2, and transmitted into the

ICR cell where they were irradiated with electrons emitted from an indirectly heated dispenser cathode (Heatwave, Watsonville, CA, USA) for ion fragmentation. The following ECD and EID parameters were employed: electron irradiation time between 35 and 100 ms, cathode potential at 0.2 - 1.2 V (ECD), or at 17 – 27 V (EID).

2.3 Results and Discussion

2.3.1 Distinguishing the isomers

ESI FTICR MS of the trypsin digested A β 1-40

(DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV, hereafter abbreviated as

Aβ40) revealed good ionization efficiency of the peptide 17-28 (Figure 2.2.a,

LVFFAEDVGSNK, abbreviated as A β17-28). The doubly charged precursor ion of this peptide was isolated, accumulated, and irradiated with low energy electrons for the ECD event as described in the experimental section. ECD mass spectra of this peptide showed abundant fragmentation with 95 % sequence coverage (Figure 2.2.a). In addition, specific neutral losses from the reduced species of the doubly-charged tryptic

180 peptide were observed, similar to those detected earlier: the loss of (C 3H4O2+NH 3)

from glutamic acid, (C 3H7+NH 3) from leucine, CH 3NO from asparagine, and C 2H4O2 loss

from aspartic acid, as well as NH 3 from the N-terminus.

Figure 2.2 ECD of Aβ17-28 (a), ECD of isoAβ17-28 (b), and EID of isoAβ17-28 (c).

In order to perform a comparative analysis of peptide isomers, the same A β 17-

28 peptide was custom-synthesized with isoaspartic acid at position 23 [isoD23] (Figure

2.2.b, LVFFAE isoD VGSNK, abbreviated as isoA β17-28). ESI FTICR MS spectra of the

isolated doubly charged A β17-28 and isoA β17-28 precursor ions showed identical m/z

• (data not shown). ECD of isoA β17-28 generated the z6 -57 diagnostic fragment ion

which was not found in the A β17-28 peptide ECD spectrum. In contrast, carbon dioxide

• (CO 2, m/z 43.9898) was lost from the same z6 fragment ion of A β17-28, but not from

isoA β17-28 (Figure 2.2.a, b insets), indicating that CO 2 loss is diagnostic for the presence of Asp, but not isoAsp at a particular residue. While the utility of CO 2 loss as a

diagnostic ion has been previously shown, 178 it is not normally as reliable as the

cn+57 /z m-n-57 diagnostic peaks as isoAsp can also lose CO 2. However, loss of CO 2 from

isoAsp appears to be a minor dissociation channel (as evident by the small peak in a

178 • 100 % isoAsp-containing peptide ). In the current study, loss of CO 2 from z 6 is not

• observed (<1 % relative abundance) in isoA β17-28 indicating that z6 -44 is another

diagnostic fragment ion for Asp-containing A β peptides. A nominal loss of 60 was also

detected from the charge reduced molecular ion species of the isomerized peptide, but

its exact mass corresponds to the leucine side chain plus ammonium (60.0813,

C3H7+NH 3), not the sidechain of aspartic acid (60.0211, C2H4O2), indicating that aspartic

acid was not present in the modified peptide. Interestingly, as shown in the inset of the

• c10 radical fragment, this ion was of a lower abundance in the isomerized version of the

peptide. This could be explained by the fact that isoaspartic acid has a shorter side

chain, which may affect hydrogen bond formation, and the hydrogen transfer within the

complex of c and z• fragments. 53, 69, 70, 181, 182 It could be assumed that a weaker

hydrogen bonded complex will have a shorter lifetime, leading to a lower degree of

intracomplex hydrogen atom transfer ( c + z• / c• + z ), and fewer number of c• radicals.

Furthermore, the fragmentation pattern of the isoA β17-28 peptide produced by

EID was also investigated to determine whether it could be used to establish the presence and location of an isoaspartic acid. EID spectrum of the isoA β17-28 contained many a, b and y, as well as c and z• type fragments (Figure 2.2.c). The z•-57 diagnostic

fragment was also present in the spectrum (with an error of 1.2 ppm, indicating a

confident assignment), although with lower abundance compared to the ECD spectra,

correlating with a general reduction in abundance of all c and z• type fragments as

competing reaction channels were accessed. Nevertheless, this experiment

demonstrated that EID can be applied in isoaspartomics research along with ECD. The

EID technique will be particularly useful in the analysis of singly charged molecular ions,

when small molecules are studied in ESI or when MALDI is used as the ionization

technique.

Generally, the isomers were distinguished by means of ECD and EID using the

•+ • • diagnostic ions [M+2H-60] and z6 -44 when aspartic acid and z6 -57 when isoaspartic

acid were present.

2.3.2 ECD of the full length Amyloid beta protein fragment 1-40

The analysis of A β40 is rather challenging due to its high hydrophobicity at its C-

terminus and its tendency to aggregate. The ESI spectrum of its tryptic digest showed

only one peptide A β17-28 with high abundance (data not shown). The other three tryptic

peptides were either suppressed or had lower ionization efficiency, which made it difficult

to isolate them for MS/MS analysis. In addition, top-down analysis of A β is

advantageous because digestion solutions are usually of elevated pH relative to

physiological conditions, which can introduce undesired structural changes as well as

additional deamidation and isomerization. 109, 116, 183 In biological fluids or tissues,

Amyloid beta is present as a mixture of various length fragments from 13 to 42 amino

acids long. 184 Digestion of this mixture will complicate the analysis and will create biases in the identification of the origins of the resulting digest peptides. Thus, it is important to create a top-down approach for the analysis of A β peptides. At this stage of the

research, an entire A β40 peptide was electrosprayed into the mass spectrometer and

analyzed by ECD. The 3 + to 5 + charged ions were observed in the spectra and the

highest charge state [M+5H] 5+ ions were isolated for ECD analysis. The ECD spectrum

of A β40 showed extensive fragmentation (Figure 2.3). Insets show the charge-reduced

species [M+5H] 4+• and [M+5H] 3+•• , as well as their neutral losses. Importantly, the

• • complementary fragment pairs c6/z 34 , and c22/ z18 adjacent to aspartic acids at positions

6 and 23 respectively are present in the spectra. It is likely that these fragment ions

would generate diagnostic fragments in isoAsp containing peptides, which could be used

to determine the presence of isoaspartic acid.

Figure 2.3 ECD of A β40.

2.3.3 Detecting isoAsp in Amyloid beta 1-42

The amyloid beta fragment 1-42, or Aβ42, which includes 2 more hydrophobic amino acids at the C-terminus compared to A β40 DAE…GAIIGLMVGGVVIA 42 , is the

most abundant in plaque deposits. Its level is greatly enhanced in AD brain, and it is

believed to be the most toxic of all Aβ peptides. 185, 186 It is worthwhile, therefore, to

develop a method for the analysis of this particular fragment, even though it is

notoriously difficult to analyze due to its high hydrophobicity and tendency to aggregate.

In addition, longer peptides generally have higher probability for significant secondary

structure in the gas phase and form numerous hydrogen bonds. Unless such ions are

activated prior to ECD analysis to disrupt secondary structure, strong hydrogen bonds

tend to keep c/z • fragment pairs in a complex, reducing the net efficiency of ECD

analysis. Nevertheless, successful analysis of A β42 would create a method suitable for

different length A β peptides with the same or fewer number of amino acids.

Formation of isoaspartic acid in a standard A β42 was induced to create a

relevant test-case for development of this method. Because the rate of aspartate

isomerization is ~40 times slower than the rate of Asn deamidation, 109 the variant

containing Japanese –Tottori mutation [Asn7] was chosen to accelerate formation of

isoAsp by deamidating the asparagine residue. The sample was incubated in

ammonium hydroxide at pH >10.2, overnight at 37°C a nd analyzed in ESI FTICR MS

using ECD. [M+5H] 5+ molecular ions were isolated, accumulated and irradiated with low-energy electrons. An ECD spectrum of the peptide is shown in Figure 2.4. The mass of the parent ion increased by one or two units (de-convoluted mass) indicating the presence of single and double deamidation correspondingly (see insets). Double deamidation was observed due to the presence of a second asparagine in the peptide sequence at position 27 . Partial deamidation of Asn27 was also observed, which is

explained by the fact that the rate of HNS deamidation is faster than the rate of SNK

deamidation. In particular, according to the experimental data acquired at near-

physiological conditions, the first order deamidation half-life of G HNS G and G SNK G model pentapeptides are 15.7 and 55.5 days. 112 For the purpose of this experiment,

however, deamidation of Asn27 was of a lesser interest, as was the completeness of the

deamidation reaction in general. Deamidation of asparagine [Asn7] was performed to

induce the formation of isoaspartic acid at this particular residue because in Amyloid beta peptides found in the cerebral plaque samples of Alzheimer patients, aspartic acid is 75 % isomerized to isoAsp at the residue position 7. 119 As a result of ECD analysis of the deamidated A β42 peptide (Figure 2.4 upper spectrum), the diagnostic fragment c6+57 was observed (at 2.2 ppm mass accuracy), demonstrating the formation of isoaspartic acid at the 7th residue and the ability of the current method to detect and localize the modification. It should be noted that this model system experienced a highly non-physiological (pH >10) environment, so that the deamidation mechanism is unlikely to follow the in vivo mechanism as the pH would have been likely to unfold the peptide, thus perturbing the native deamidation rate. However, the results of this experiment provide a needed baseline for future studies. It was clearly demonstrated that ECD can be used to detect the isoAsp in a top-down experiment of the longest A β peptide.

Figure 2.4 ECD of A β42 deamidated at Asn7 and Asn27.

Exploring these studies further in terms of quantitative analysis and combining this technique together with HPLC separation will present a powerful tool for the analysis of physiological fluids in Alzheimer’s disease patients. Comparative studies of isoAsp containing A β peptides in normal and diseased samples of different stages might reveal a novel perspective on Alzheimer’s disease initiation and progression.

2.4 Conclusion

•+ • The ECD diagnostic ions [M+2H-60] and z6 -57 were observed and successfully

applied to differentiate the isomeric forms of the Amyloid β tryptic peptide 17-28.

Differentiation of aspartic and isoaspartic acid residues is also possible using EID using

• the same diagnostic ion z6 -57 as in ECD. Although the abundance of the diagnostic peak was relatively low compare to ECD, EID could be particularly useful in the analysis of singly charged precursor ions, where ECD is not applicable. Amyloid β 1-40 and 1-42 peptides can be analyzed by a top-down ECD approach without prior digestion and provide substantial sequence coverage. Isomerized Amyloid β 1-42 at the residue 7 can be differentiated by ECD using the c6+57 diagnostic ion. Both ECD and EID can clearly

define the presence and the position of isoaspartic acid residues in Amyloid β peptides;

therefore, they could be applied to the samples of Alzheimer’s patients or healthy

individuals for better understanding of the disease.

Chapter 3

The analysis of isoAsp, Asp, and Asn containing peptide variants by Liquid

Chromatography

3.1 Introduction

The great challenge of biological sample analysis is the complexity of the sample. Often, PTM-containing species represent the minority of the mixture and are easily neglected or lost. Thus, separation of the complex sample becomes crucial for its analysis. High performance liquid chromatography (HPLC) is a powerful technique for the separation of biological mixtures. It was shown in the previous chapter that isoAsp containing standard peptides and proteins can be unambiguously identified using ExD tandem MS methods. However, when isoAsp, Asp and Asn are present as a mixture, which is most often the case in a real biological sample, the abundance of diagnostic fragment is diluted in the spectrum and could be neglected or not observed during analysis. Thus, in many instances, it is important to perform sample separation for

“enrichment” of the peptides containing the modification. Further, when ExD methods are not available to differentiate Asp and isoAsp residues in peptides, distinguishing between the two modifications is often based on the assumption that peptides with isoAsp residue normally elute earlier compared with their non-modified counterparts in

RP-HPLC. 165, 187-189 However, this is not always the case and such assignment must be

checked. 170, 171, 190, 191 Thus, in this chapter, HPLC separation is applied to study isoAsp,

______1This chapter has been partially reproduced with permission from Sargaeva, N. P.; Goloborodko, A. A.; Moskovets, E.; Gorshkov, M. V.; O'Connor, P. B., Sequence-specific predictive chromatography to assist mass spectrometric analysis of asparagine deamidation and aspartate isomerization in peptides, Electrophoresis , 2011 , 32 , 1962-1969. 74

Asp, and Asn containing model peptides to better understand their elution behavior.

These include several groups of peptides containing Asn deamidation and Asp

isomerization sites at various positions within the peptide sequence. The change of the

ion paring reagent in the mobile phase is tested on peptide elution as well. These data

are compared to the results provided by other research groups in the summary table.

The second part of this chapter focuses on predictive chromatography. The

retention time prediction is commonly used to predict the elution times of non-modified

peptides to improve their identification. 192, 193 Recently, a novel model was introduced to

describe separation of peptides under reversed phase (RP) high performance liquid

chromatography (HPLC) conditions based on a concept of liquid chromatography at

critical conditions (BioLCCC, Institute of Energy problems of Chemical Physics in

Moscow, Russia). 194-196 The BioLCCC model considers the thermodynamic aspects of peptide separation in reversed-phases. Contrary to existing empirical models, it uses a limited number of free phenomenological parameters and is inherently sequence dependent. 197 The calibration of the BioLCCC model necessary to predict retention

times (RT) in the RP-LC separation of unmodified peptides uses only 24 specially

designed synthetic peptides. A new module accounting for presence of a

phosphogroup 198 and isoAsp/Asp modifications 191 has been added very recently to the

BioLCCC predictor. When extended to predict RTs in RP-LC separation of peptides with

modified residues, the BioLCCC model, due to its inherent sequence dependence, is

capable of predicting RTs of peptides differing only by location of modified amino acids.

Therefore, based on the above results of the RP-HPLC separation of peptides with Asn,

Asp, and isoAsp residues, the feasibility of using the BioLCCC model to accurately

describe their retention times is tested.

3.2 Experimental section

3.2.1 Peptides and reagents

The following peptides were custom synthesized by Peptide 2.0 (Chantilly, VA,

USA): yeast enolase peptide variants H-IGL NCASSEFFK-OH, H-IGL iD CASSEFFK-OH,

H-IGL DCASSEFFK-OH, synthetic peptides Ac-NGVG NVGGVH-NH 2, Ac- isoD GVG isoD VGGVH-NH 2, Ac-DGVG isoD VGGVH-NH 2, and Ac-DGVG DVGGVH-NH 2.

Angiotensin II (AngII) peptide variants H-DRVYIHPF-OH, H-NRVYIHPF-OH, and H- isoD RVYIHPF-OH were obtained from AnaSpec (San Jose, CA, USA). Trifluoroacetic

Acid (TFA) and Formic Acid (FA) were purchased from Thermo Scientific (Rockford, IL,

USA); HPLC grade Acetonitrile (ACN) and Methanol (MeOH) were purchased from

Fisher Scientific (Fair Lawn, NJ, USA); deionized water was purified by a Millipore Milli-Q

Gradient system (R=18.2 M Ω cm and TOC=9-12 ppb), (Billerica, MA, USA); Ammonium

Hydroxide (20 %) was obtained from JT Baker (Phillipsburg, NJ, USA), and Ammonium

Bicarbonate (ABC) from Sigma-Aldrich, (St. Louis, MO, USA).

3.2.2 Liquid Chromatography

Reversed-phase high performance liquid chromatography peptide separations were performed using an Agilent 1200 Series system (Agilent Technologies, Wilmington,

DE, USA) and reversed-phase C18 columns Vydac 218TP54 C18 (250x4.6mm, 5µm particles, 300Å pore size) and Vydac 218TP5215 C18 (150x2.1mm, 3µm particles, 300Å pore size). 20 µl of 0.5-4 nmol peptide mixtures were injected directly onto the column and eluted with various gradients of acetonitrile with either 0.08 % TFA or 0.1 % FA at

0.5-1 ml/min flow rate and 35 °C unless indicated o therwise. These TFA and FA concentrations correspond to pH 2.0 and 2.6, respectively. A linear or step gradient was adjusted for each group of peptides. The chromatograms were measured using UV detection at 214 nm and the fractions were collected and dried in a SpeedVac (when eluted on TFA) or refrigerated at 4 °C (when eluted on FA) for further analysis.

3.2.3 Mass spectrometry

ECD-FTICR-MS. Electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry was performed on a 12 T SolariX FTICR MS instrument

(Bruker Daltonics, Billerica, MA, USA). Peptides were nanosprayed at a typical concentration of 0.5-2 µM in ACN:H 2O (0.1 % FA) when mass analysis was conducted following HPLC separation with off-line fraction collection or in 50:50 MeOH: H 2O with

(0.1 % FA) when infused directly. Doubly charged molecular ions were isolated and irradiated with low-energy (<1 eV) electrons for 0.05-0.08 ms to produce fragments. 100 scans were accumulated for each peptide and spectra were analyzed using Bruker’s ESI

Compass DataAnalysis 4.0 software.

3.2.4 RT prediction

Theoretical retention times for peptides were calculated using the BioLCCC model for sequence-dependent RT prediction. This software package was developed as an open source library and is available on-line at http://theorchromo.ru . It consists of a library written in both C++ and Python programming languages and allows setting the user-defined separation conditions including the choice of ion-pairing reagents, column parameters, and the gradient profile.

3.3 Results and Discussion

3.3.1 RP-HPLC separations of isoAsp-peptides

Yeast enolase model peptide variants containing Asp, isoAsp and Asn in the middle of the sequence are first separated using a mobile phase with FA as the ion paring reagent and then compared with that with TFA (Figure 3.1.a and 3.1.b respectively). The elution order in both scenarios is isoAsp <= Asn < Asp, although with

FA, there is no baseline separation observed for isoAsp/Asn variants, and isoAsp tends to elute first. Nonetheless, the isoAsp elution prior to the Asp variant is consistent with the results reported earlier. 165, 187-189

Figure 3.1 IsoAsp position affects the peptide elution order.

The next group of peptide variants of Ang II contains modification at the peptide

N-terminal site. A sudden change is observed in the elution order of FA separated peptides: Asn < Asp < isoAsp, the isoAsp-peptide eluted last, however, Asn-, and Asp- peptides kept the order (Figure 3.1.c). The Ang II peptides are further separated with alternative ion paring reagent TFA and the isoAsp-peptide retention time is shifted again

(Figure 3.1.d). The elution order is as follows: Asn < isoAsp < Asp. These results indicate that isoAsp position within the peptide sequence plays an important role in the hydrophobic interaction between the peptide and the column’s stationary phase. The difference in the isoAsp-peptide elution order when different ion paring reagents applied

may be related to the difference in pH of the mobile phase: 2.6 (FA) and 2.0 (TFA).

Although the difference is very small, the pKa value of an isoAsp residue (~3.3) is lower

than that of an Asp residue (~4). 153 Therefore, at more acidic conditions such as in

TFA, an isoAsp-peptide gets higher rate of deprotonation weakening the hydrophobic

interaction, leading to a shorter retention time. Interestingly, this change is only

observed when the isoAsp is positioned at the N-terminus, so it seems that for small

peptides the charge of the N-terminus plays a very important role in peptide partitioning

between the mobile phase and the RP column.

Figure 3.2 Chromatograms of Ang II peptide variants.

It must be noted that chromatogram assignment is based on multiple separations

(>2) for each peptide group and at first each model peptide was loaded onto the column by itself to obtain the reference retention time information. An example is shown for

Ang II peptide in Figure 3.2. In addition, fractions were collected for each group of peptides when loaded as a mixture onto the column and sequence information was confirmed by ECD (data will be presented in the following chapter).

To further test the importance of the isoAsp position within the peptide, the next group of synthetic peptides is analyzed. In this case, the N-terminal isoAsp is capped with an acetyl group. There is also an additional modification site present in the middle of the sequence. Peptides were separated at the same conditions with either an FA or a

TFA pH modifier. Corresponding chromatograms and sequences of peptides are presented in Figure 3.3.a and Figure 3.3.b. This time, the elution order has not changed with the change of the ion paring reagent. Overall, the elution behavior of this group of peptides resembled that of the yeast enolase peptide variants (Figure 3.1.a, b).

Therefore, it seems that acetylation of the N-terminal isoAsp preserves the elution order of peptide variants.

Figure 3.3 N-terminal acetylation preserves the elution order of isoAsp modified peptides.

Table 3.1 The elution order results for peptides containing Asn, Asp, and isoAsp residues.

Table 3.1 summarizes the obtained results and results reported on similar peptides in earlier works.170, 171, 187, 190 In general, the use of TFA results in isoAsp-

containing peptides eluting before the Asp-containing ones. The same is applicable to peptides with isoAsp located in the middle of the peptide sequence and separated using

FA; however, this is not always the case as was shown for Cytochrome -C and A β 1-16 peptides studied by Ni, W., et al. 171 Additionally, based on the results of Ang II peptides

(Figure 3.1), when isoAsp is located at the non-acetylated N-terminus (i.e. the one with normal composition), the observed retention time is increased, and the elution order is reversed: Asp < isoAsp. This was also demonstrated earlier on the DQPIASTK peptide variants by Winter et al. (Table 3.1). 170 The reversed order of the N-terminal isoAsp containing peptides may be attributed to the presence of the charged N-terminal group, which may be overcome by acetylation of the N-terminus as shown in Figure 3.2.

In conclusion of this section, it seems clear that when dealing with peptides with deamidation and isomerization modifications, the LC separation results alone may lead to erroneous assignment, because the elution order depends on the peptide sequence as well as on conditions of separation. Additional methods such as ExD tandem MS is a necessary complementary technique to validate the assignment.

3.3.2 Predictive chromatography

When ExD tandem MS is not available, other means of complementary analysis would be desired for correct identification of the isoAsp-peptides. One such tool is predictive chromatography. The retention time prediction is offered as an additional step of the analysis in the standard proteomic approach of peptide sequence identification

(Figure 3.4). Information from the database search and the HPLC parameters could be used to predict the retention time of the eluted peptides. Thus, for instance, the results may be corrected to reduce the false positive identifications from the database outcome.

Figure 3.4 Improved sequence identification (ID)

One analytical model of retention time prediction is the BioLCCC model developed at the

Institute of Energy problems of Chemical Physics in Moscow, Russia for non-modified peptides. 194 This model takes into account column dimensions (length, i.d.), packing material (pore size), mobile phase components, gradient details, flow rate, and peptide sequence. It is possible to extend this model toward modified peptides 196 ; the model was recently extended to take into account the separation of peptides containing isoAsp residues. As a result of a collaborative project, this model is tested on modified peptide separations presented above. Ang II peptide variants separation in FA (Figure 3.5.a) provided perfect correlation between the predicted theoretical and the experimental data.

RT was also calculated for a group of peptides separated in TFA as demonstrated for synthetic peptides (Figure 3.5.c, d). Correlation between the theoretical RT and the experimental RT is also near 100 %. Notably, when predicting peptide elution orders in

FA vs. TFA, the model had to be recalibrated to address separation selectivity change.

The results shown in Figure 3.5 demonstrate that the BioLCCC model describing the separation of peptides in reversed-phase gradient HPLC can be used to predict elution times and elution order for peptides containing Asn, Asp and isoAsp residues. In the course of protein analysis that requires identification of localization of the modified residues and characterization of the level of protein deamidation or isomerization, the sequence specific prediction of retention times for such peptide variants will provide an additional set of sequence data complementary to MS/MS results as suggested in Figure

3.6. The use of retention time prediction specific to amino acid sequence, may assist in

characterization of proteins with isomeric amino acid residues in a variety of

applications. These applications may include selected reaction monitoring (SRM) or

multiple reaction monitoring (MRM) analysis in targeted proteomics, which are

increasingly used in biomarker discovery. The match between observed and predicted

retention times can serve as an independent validation of identifications of peptides with

PTM in large-scale proteomic projects.

Figure 3.6 Improved identification of modified peptides.

3.4 Conclusion

In this chapter, it is demonstrated that chromatographic results alone may be biased especially when N-terminal isoAsp is present. The ExD methods would be required to validate or correct isomer assignment. When ExD methods are not available, the use of other complementary techniques is needed. Predictive chromatography was suggested as one such tool. Indeed, it was shown that sequence specific retention time predicting model, the BioLCCC, is capable of providing correct

elution orders for isoAsp-containing peptides. Matching of the predicted and experimental retention times allows additional validation of peptide identifications obtained from MS/MS spectra and filtering out possible false positives due to sequence specificity of chromatographic data. It is worth noting, that any predictor model, such as the one described above, needs to be established and validated by employing a vast variety of experimental data. While mathematical predictor models can significantly aid in experimental design and outcome, it is desirable to validate the data and the data assumption by obtaining empirical data.

Chapter 4

Differentiating N-terminal Asp and isoAsp residues in peptides

4.1 Introduction

Formation of isoaspartic acid (isoAsp) is a common modification of aspartic acid

(Asp) or asparagine (Asn) residue in proteins. Differentiation of isoAsp and Asp

residues is a challenging task owing to their similar properties and identical molecular

mass. A number of techniques have been developed to detect isoAsp, and they are

often used in conjunction. These include the Edman degradation reaction, 199 PIMT-

utilizing assays, 154 affinity enrichment of isoAsp-containing proteins,175 use of isoAsp-

specific antibodies, 120, 158 endoproteinase Asp-N-based approaches, 125, 127, 171 isotopic

18 O labeling, 111, 173, 183 and various liquid chromatography (LC)-based techniques:

hydrophobic interaction chromatography, 200 size-exclusion high performance liquid

chromatography (HPLC), 125 cation exchange HPLC, 200 Reversed-Phase (RP) HPLC, 168,

190 and Ultra HPLC (UPLC). 170 Asp/isoAsp differentiation has been demonstrated by

tandem mass spectrometric approaches including collisionally activated dissociation

(CAD),162, 164 fast atom bombardment, 163 and post-source decay fragmentation. 201

Recently, isoAsp identification and quantitation by tandem MS methods employing ion-

electron or ion-ion interactions (ExD) have been developed 176, 178, 202-204 and successfully

applied, 171, 205, 206 based on the diagnostic fragment ions c + 57 and z• - 57 that result from the C α - Cβ bond cleavage unique to isoAsp residues. All the above-mentioned MS

______1This chapter has been partially reproduced with permission from Sargaeva, N. P.; Lin, C.; O'Connor, P. B., Differentiating N-terminal aspartic and isoaspartic acid residues in peptides, Analytical Chemistry , 2011 , 83 , 6675-6682. 88

methods were accomplished in the positive ion mode; however, in the negative ion

mode, the isoAsp-containing peptides were not differentiated. 207

In spite of a large number of existing approaches, isoAsp identification remains a

challenging task. In HPLC, the elution order of Asp/isoAsp-containing peptides depends

on conditions of separation (column type, mobile phase, temperature, etc.), materials,

and the instrument used. 171, 190 In particular, as was shown in the previous chapter, the elution order can be inverted when isoAsp is located at the N-terminus, leading to erroneous assignment. 170, 208 Thus, unless a standard mixture of the same peptide pair

separated at the same condition is available, a further analysis would be required for

correct isomer assignment. ExD-based tandem MS seems to be a fast and accurate

approach well suited for this task. However, to the best of author knowledge, a detailed

ExD study of peptides with N-terminally located isoAsp residues has not been performed

and the N-terminal isoAsp-specific fragments have not yet been demonstrated. In this

chapter, the potential of ExD for N-terminal isoAsp residue identification is investigated,

either by performing the ExD analysis alone or following RP-HPLC separation. The role

of N-terminal acetylation and the effect of ion activation in isomer differentiation are also

examined.

4.2 Experimental section

4.2.1 Peptides and reagents

Angiotensin II (Ang II) peptide variant i DRVYIHPF (hereinafter iD represents isoAsp in peptide sequences), synthetic peptides Ac-DGVG DVGGVH-NH 2 and

Ac-iD GVG iD VGGVH-NH 2, and Amyloid beta 1-10 peptide variant NAEFRH NSGY with

Asn residues at position 1 and 7 (hereafter A β1-10 (N1N7)) were custom synthesized by

- Peptide 2.0 (Chantilly, VA, USA). Ang II ( DRVYIHPF) and A β1 10 ( DAEFRH DSGY) were

ordered from AnaSpec (San Jose, CA, USA). Formic Acid (FA) was purchased from

Thermo Scientific (Rockford, IL, USA). HPLC grade Acetonitrile (ACN) and Methanol

(MeOH) were obtained from Fisher Scientific (Fair Lawn, NJ, USA). Deionized water

was purified by a Millipore Milli-Q Gradient system (R=18.2 MΩ cm and TOC = 9 –

12 ppb) (Billerica, MA, USA). Ammonium Bicarbonate (ABC) was ordered from Sigma-

Aldrich (St. Louis, MO, USA).

4.2.2 Deamidation

The A β1-10 peptide variant (NAEFRHNSGY) was incubated at 0.44 mM

concentration in 0.1 mM ABC, at pH ~7.7 and 37 °C f or 4 days. The resulting peptide

mixture was separated on reversed-phase C 18 column prior to MS/MS analysis.

4.2.3 Chromatography

RP-HPLC peptide separations were performed on an Agilent 1200 Series system

(Agilent Technologies, Wilmington, DE, USA) using a reversed-phase C18 column

Vydac 218TP5215 (150 x 2.1 mm, 3 µm particles, 300 Å pore size). 4 nmol of peptide mixture (in 20 µl) was injected directly into the column and eluted using a linear gradient of acetonitrile with 0.1 % FA (0 min - 1 % B, 20 min - 11 % B) at 0.7 ml/min flow rate at

60 °C. The chromatograms were measured using UV de tection at 214 nm. Fractions were collected and refrigerated at 4 °C prior to MS /MS analysis.

4.2.4 Mass Spectrometry

ExD analyses were performed on:

1) a solariX FTICR instrument (Bruker Daltonics, Billerica, MA, USA) with a 12 T actively shielded magnet: electron capture dissociation (ECD), hotECD, and electron transfer dissociation (ETD). Peptides were nanosprayed with 0.5 - 2 µM concentration in

50:50 ACN:H 2O with 0.1 % FA following HPLC separation with off-line fraction collection or in 50:50 MeOH:H 2O with 0.1 % FA when infused directly. Doubly-charged molecular

ions were isolated and irradiated with low- (cathode bias 0.4-0.8 V) or high- (cathode

bias 4.5 V (hotECD)) energy electrons for 0.05 - 0.5 ms to produce fragments. Each

ExD spectrum was the result of 100 scans.

2) an LTQ-Orbitrap XL with ETD capability (Thermo Scientific, San Jose, CA,

USA): ETD with or without supplemental activation (SA). Peptides were nanosprayed in

solutions as stated above at ~ 0.5 µM using a robotic Nanomate source (Advion, Ithaca,

NY, USA). Activation energy parameter “En” of 5 or 15 was applied when SA was on

(typical En values range from 0 - 20).

and 3) an amaZon Ion Trap instrument (Bruker Daltonics, Billerica, MA, USA):

ETD with or without smart decomposition (SD). Peptides were electrosprayed (2 µl/min,

glass capillary temperature 220 °C) at 1 µM concent ration in 50:50 MeOH:H2O with

0.1-1 % formic acid using an Apollo II ion source.

Fluoranthene was used as the anion radical reagent in all ETD experiments with

150 - 250 ms reagent accumulation, and 50 - 150 ms reaction time was used to produce fragments. Data acquired on solariX and amaZon were analyzed using Bruker’s ESI

Compass DataAnalysis 4.0 software. The Orbitrap data were analyzed using Thermo's

Xcalibur 2.0.7 software.

4.3 Results and Discussion

4.3.1 Diagnostic fragments for the N-terminal Asp/isoAsp

As originally proposed, ECD of peptides with isoAsp located at the nth position

• may generate a unique complementary ion pair cn-1 + 57 and zm- n + 1 - 57 as a result of the C α - Cβ bond cleavage, where m is the total number of amino acid residues and 57,

• or more accurately, 56.9976 corresponds to the mass of a C 2HO 2 group. For an isoAsp

residue located at the N-terminus, n = 1, and the diagnostic fragments become c0 + 57

• and zm - 57. The c0 fragment is essentially an ammonia molecule which is neither

• informative nor observed in ECD spectra, and the zm ion contains no sequence

information, and is usually assigned as a loss of ammonia from the charge-reduced

+• species [M + 2H - NH 3] . Thus, when an N-terminal isoAsp residue is present, the C-

• +• terminal diagnostic fragment would be [M + 2H - (NH3 + C2HO 2 )] or

[M + 2H -74.0242] +• (hereinafter indicated as [M + 2H - 74] +• ), and the N-terminal

+• diagnostic fragment would be C 2H5NO 2 (m/z = 75.032). Although the m/z = 75 peak is

usually below the low mass cut-off, it is still possible to look for the corresponding neutral

loss [M + 2H - 74] +• .

In order to see if the isoAsp can be differentiated from the Asp when located at

the N-terminus, ECD spectra of the two synthetic isomers of Ang II were directly

compared (Figure 4.1). The two spectra were very similar with only a few exceptions.

• Careful examination revealed the presence of the specific fragment “ z8 ” - 57 or

[M + 2H - 74] +• only in the ECD spectrum of the isoAsp-containing Ang II peptide. In

contrast, the loss of 60.0211 (C 2H4O2) and a double CO 2 loss were observed only for the

Asp peptide. Indeed, the loss of 60 is characteristic of the Asp side chain 180 and cannot

be formed from the isoAsp. The loss of two CO 2 molecules can be generated by a

combined loss from the C-terminus and from the Asp side chain. Although the CO 2 loss

can also come from an isoAsp residue, it is normally of a much lower abundance 178, 205 or

202 • undetectable. For example, the a6 - NH 2 - CO 2 peak was very abundant in the ECD

spectrum of the Ang II peptide, but was not detected in that of its isoAsp variant. Thus,

in Figure 4.1a, the CO 2 loss from the charge-reduced species was probably generated

from the C-terminus.

Figure 4.1 ECD spectra of the Angiotensin II (a) and its modified variant with isoAsp at the N-terminus (b). Insets show side-chain losses from the charge-reduced species

+• +• [M + 2H] . IsoAsp specific fragment [M + 2H - 74] , produced as a result of the C α - Cβ bond cleavage within the N-terminal isoAsp residue, is present in the modified variant only, (b) inset.

4.3.2 Analysis of the deamidation products of A β1-10 (N1N7) by off-line RP-HPLC followed by ECD

An A β1-10 peptide variant with Asn residues at position 1 and 7 was deamidated to artificially introduce isoAsp residues into the sequence. The resulting mixture of peptides was separated on a C 18 reversed-phase column. 11 peaks were detected in

the chromatogram (Figure 4.2, top panel), collected into fractions, and further analyzed

using ECD. It was possible to differentiate and to define the elution order of the isomeric

peptides containing isoAsp residues based on the diagnostic fragments observed, i.e.

+• [M + 2H - 74] for the isoAsp1 and c6 + 57 for the isoAsp7, as shown in Figure 4.2. Non- deamidated Asn residues were distinguished based on the 0.984 mass difference of the

• precursor ions and their fragments containing Asn, as shown in the example of z6

fragments (Figure 4.3. fractions I and V). Fraction VI contained both peptides

DAEFRH NSGY and NAEFRH iD SGY, which are isomers and thus indistinguishable at

the MS level yet could be easily identified by ECD because some fragment ions

exhibited two components, separated by 0.984 Da (Figure 4.3. fraction VI, inset). The

DAEFRHNSGY peptide comprises only a small fraction of fraction VI, based on the

• relative abundance of the two components within the z6 isotopic cluster. This is

consistent with the fact that the DAEFRH NSGY peptide has a lower probability of formation comparing to the NAEFRH iD SGY peptide (from original peptide

NAEFRH NSGY) because the Asn in the NA sequence has a slower deamidation rate than Asn in the NS sequence. 115, 209 Spectra for fractions II and III are not shown as they consist of unrelated peptides originated from synthesis impurity. Although the separation of this closely related mixture of peptides was challenging and for some peaks there was no base line separation, all peptides were successfully identified by ECD, highlighting the utility of ECD in isoAspartomic research.

Figure 4.2 Chromatogram of the peptide mixture, generated by partial deamidation of the A β1-10 (N1N7) peptide (top panel). Each chroma tographic peak labeled based on

ECD results. ECD spectra shown correspond to the VIII-XI fractions with the isoAsp specific fragments indicated in the insets.

Figure 4.3. ECD spectra of chromatographic peaks I, IV-VII from the partially deamidated A β1-10 (N1N7) peptide mixture.

4.3.3 Peak interference

Caution should be taken if the peptide of interest contains methionine (Met) or

isoleucine (Ile) residues because they may produce side-chain loss fragment ions with

m/z close to that of the diagnostic fragment ion: the Met side-chain loss (C 3H6S) results in a peak at [M + 2H - 74.01902] +• , and the Ile side-chain loss with an additional

+• ammonia loss (C 4H8 + NH 3) at [M + 2H - 73.0891] . Although these interfering peaks

could lead to erroneous identification of N-terminal isoAsp, they do not usually pose a

problem if the analysis is performed on an instrument with sufficient resolving power.

For example, differentiating the singly-charged N-terminal isoAsp signature fragment ion

at m/z = 1000 from the interfering Met side-chain loss ion would require a mass resolving

power of > 190,000, which is easily achievable on an FTICR mass spectrometer such as

the one used here.

4.3.4 N-terminal acetylation

For experiments conducted on instruments with limited mass resolving power,

such as ETD analysis done on an ion trap instrument, the peak interference problem

could be circumvented by shifting the diagnostic peak out of the small molecule and

side-chain loss region. This can be done by, for example, N-terminal acetylation. In this

case, the C-terminal diagnostic fragment would be

• +• +• [M + 2H - (C 2H5NO + C2HO 2 )] = [M + 2H - 116.0348] (hereinafter indicated as

+• +• [M + 2H - 116] ), and the N-terminal diagnostic fragment would be C 4H7NO 3 with

m/z of 117.042. The effect of N-terminal acetylation was examined on the synthetic

peptide Ac-DGVGDVGGVH-NH 2 and its isoAsp-variant Ac-iDGVGiDVGGVH-NH 2 using

ECD. Specific diagnostic fragment peaks were observed for both isomers (Figure 4.4,

insets): [M + 2H - 116] +• was present exclusively in the isoAsp-peptide spectrum, and

the neutral loss of 60 (C 2H4O2 = 60.0211) was only observed from the charge-reduced

species of the Asp-peptide variant, indicated as [M + 2H - 60] +• . The peak at

m/z = 894.4195 in the isoAsp-peptide spectrum (Figure 4.4, lower panel inset) is not

related and corresponds to the neutral loss of 59 (C 2H5NO = 59.0371) from the

acetylated N-terminus. In addition, many of the fragments containing the Asp residue of

the Asp-peptide also exhibit an accompanying loss of CO 2 (Figure 4.4, upper panel); however, there are no such losses observed in the isoAsp-peptide ECD spectrum,

• except for the z6 - CO 2. This ion was produced as a result of a secondary reaction

• following the z6 fragment ion formation via hydrogen rearrangement that only occurs

when the C α radical formed is adjacent to the isoAsp side chain. Similarly, a peak at

m/z = 832 also has the CO 2 loss, which could have been formed as a result of a

secondary reaction upon the loss of an acetamide (C2H5NO).

It should be noted that, if the N-terminally acetylated peptide contains a

• tryptophan (Trp) residue, the Trp side-chain loss of C 8H6N (116.05002) would interfere

with the isoAsp diagnostic fragment peak [M + 2H - 116.0348] +• (to correctly identify such

fragments at m/z ~ 1000, the minimum required resolving power is 65,000). In addition,

no N-terminal isoAsp diagnostic fragment ion was observed in the N-terminally

acetylated peptide, likely because of its small size and lack of a charge carrier.

Therefore, other types of N-terminal elongation could be used for a more confident

identification of the N-terminal isoAsp. Presumably, the use of larger tags may help to

avoid undesired interference from the side-chain losses and for the detection of the

N-terminal diagnostic ion.

Figure 4.4 ECD spectra of the synthetic pe ptide variants with Asp (a) and isoAsp residues (b) amidated at the C-terminus and acetylated at the N-terminus. IsoAsp1 and

+• • isoAsp5 specific fragments, [M + 2H - 116] and z6 - 57, are observed onl y for the isoAsp-peptide variant. Asp side-chain losses are also indicated, (a) inset.

4.3.5 Analysis of the N-terminal isoAsp by ETD

Peptides with N-terminal isoAsp were also analyzed using ETD. The N-terminal isoAsp diagnostic fragment peak [M + 2H - 74] +• was observed for all peptides, although in some cases, diagnostic peak detection was challenging as it exhibited low intensity.

In particular, in ETD (LTQ-Orbitrap) of isoAsp-containing A β1-10 peptide, the peak corresponding to the diagnostic fragment ion was barely observable above the noise threshold level even with the supplemental activation (Figure 4.5a, inset). In addition,

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some low-abundance peaks were barely detectable or absent in the ETD spectrum, e.g.

• • a5 , and z7 - CO 2. ECD provided 4.5 times larger S/N ratio for the diagnostic fragment peak (Figure 4.5b, inset), possibly owing to the higher energy available in the ECD process than in the ETD process. Thus, the effect of additional energy input on diagnostic ion generation was further explored by means of supplemental activation.

Figure 4.5 N-terminal isoAsp residue diagnostic fragment peak [M + 2H - 74] +• detection for the A β1-10 peptide variant ( iD AEFRH iD SGY, fraction XI in the Figure 4.4, top panel) by: (a) ETD on LTQ-Orbitrap, with supplemental activation energy parameter 15; and

(b) ECD on SolariX. Insets show diagnostic peaks and their S/N ratios.

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4.3.6 The effect of ion activation

Supplemental activation can improve fragment detection. 203, 210, 211 Thus, the effect of supplemental ion activation (via SA, hotECD, and SD) on the diagnostic fragment peak intensity was investigated for all isoAsp-peptides studied. The S/N ratios and the relative intensities (Rel. Int.) of the diagnostic ion peaks are presented in Table

4.1. At first, the Rel. Int. was calculated, but it did not reflect the true picture for some peptides, because the absolute intensity of all fragments could change significantly with ion activation. Absolute S/N ratio seemed to be a better indicator than relative intensity, for example, with the Ang II ETD spectrum acquired on the amaZon instrument, application of SD resulted in a five-fold increase in the S/N ratio of the diagnostic fragment peak, while the Rel. Int. value did not change much (Table 4.1).

Table 4.1 The effect of additional energy input on the diagnostic fragment formation.

Relative intensity (Rel. Int.) was calculated as the abso lute intensity of the diagnostic fragment divided by the sum of the absolute intensities of all c and z fragments.

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Figure 4.6 N-terminal isoAsp residue diagnostic fragment peak [M + 2H - 74] +• detection using ETD without (a) or with SA (b) for the modified Ang II peptide variant

(iD RVYIHPF). Both spectra were acquired on Orbitrap. Insets show the S/N ratio of the

[M + 2H - 74] +• peak and the appearance of additional side-chain losses upon SA (His and Tyr).

A direct comparison of the results acquired on different instruments is not possible because of the difference in multiple operating parameters, energetics of the reactions and the mass analyzers. When comparing spectra obtained using the same instrument, the results suggest that supplemental activation generally leads to higher

S/N ratios for diagnostic ions. The effect of additional energy input on fragmentation

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depends on the peptide sequence, particularly, on the presence of basic residues and

side-chain interactions. An example is shown on the ETD data acquired on an Orbitrap

instrument with or without SA for the modified Ang II peptide (Figure 4.6). Similar to the

ETD results acquired on an amaZon instrument mentioned earlier, the S/N ratio of the

diagnostic ion became 4 times greater when SA was applied (Figure 4.6b).

4.4 Conclusions

The N-terminal isoAsp residue was differentiated from Asp using ECD, hotECD, and ETD. Based on the detection of the specific fragment [M + 2H - 74] +• (isoAsp1),

+• c6 + 57 (isoAsp7), and [M + 2H - 60] (Asp1, Asp7) it was possible to differentiate a mixture of 9 peptides containing isoAsp, Asp, and Asn residues, produced upon partial deamidation of the A β1-10 (N1N7) peptide and separated by a RP-HPLC system. This result illustrates that HPLC separation followed by ExD analysis can be a powerful method for isoAsp-containing peptide mixture differentiation. Although the detection of the N-terminal diagnostic fragment peak can be challenging, it is possible to substantially improve its detection by application of supplemental ion activation. In some cases, the

S/N ratio of the signature fragment ion peak was increased by up to 6 times. Several interfering peaks may further complicate the analysis when Met or Ile amino acids are present. This can be overcome by the N-terminal elongation, with, for example, acetylation, which introduces a mass shift of the diagnostic peak to the smaller m/z region where fewer side-chain losses are available. However, if a Trp residue is present in the sequence, other types of elongation would be preferred. In addition, with a larger tag attached to the N-terminus, an N-terminal diagnostic peak may become detectable.

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Chapter 5

Detection of IsoAsp in Amyloid β Peptides From CSF

5.1 Introduction

Alzheimer’s disease (AD) is a neurodegenerative disorder of the central nervous

system with the loss of brain function leading to impaired memory, cognitive dysfunction,

and altered behavior. AD is characterized by abnormal accumulation of intraneuronal

neurofibrillary tangles of hyperphosphorylated tau protein 212 and extracellular accumulation of amyloid plaques. 213 The major components of the amyloid plaques are

the β-Amyloid peptides of 40 and 42 amino acids long (A β40 and A β42) derived from the

amyloid precursor protein. Recently, independent of the clinical diagnosis, the

biomarkers of AD were proposed – the elevated level of the 181-phosphorylated Tau

protein and the decreased level of A β42 in cerebrospinal fluid (CSF). 214, 215 CSF

occupies the space around and inside the brain and spinal cord, providing basic

mechanical and immunological protection to the brain. It is absorbed into the

bloodstream, rinsing the metabolic waste of the central nervous system and can be used

to diagnose nervous system disorders and infections. The amount of CSF at each time

in the brain is 150 ml and it is produced at a rate of 500 ml/day. CSF contains

15 - 40 mg/dL of protein, which is only 0.3 % of plasma proteins.

In addition to A β42, a number of A β peptides were detected in CSF: A β13,

Aβ14, A β15, A β16, A β17, A β19, A β20, A β30, A β33, A β34, A β37, A β38, and A β40. 184

This data along with the fact that isoAsp was detected in A β peptides of amyloid

plaques 119 suggests screening for the isoAsp in peptides present in CSF as another

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potential biomarker of the disease. HPLC-ECD and ETD MS seems to be a promising

method for fast and accurate analysis of isoAsp formation in A β peptides of CSF. This

chapter will describe the preliminary results, challenges of the proposed approach, and

future directions.

5.2 Experimental section

5.2.1 Standard peptides

Standard peptides A β40 and A β42 of 90 % and 95 % purity, respectively, were

purchased from Sigma Aldrich (St. Louis, MO, USA). The isomerized form of A β17-28

(LVFFAEisoDVGSNK) was custom synthesized by AnaSpec (San Jose, CA, USA).

Peptides were dissolved in water, aliquoted into vials, dried, and kept at -20 ◦C for immediate use.

5.2.2 Immunoprecipitation of Amyloid β peptides from human CSF

CSF immunoprecipitation (IP) was performed in a collaborator laboratory, at the

Institute of Neuroscience and Physiology, University of Gothenburg, Sweden as

previously described. 184 CSF was pooled from people with memory disorders, without a

clear AD or non-AD biomarker signature. The IP was performed using three different

Aβ-specific antibodies: 6E10, 4G8, and 11A50-B10 (Signet Laboratories, Dedham,

Mass., USA), which target epitopes 4-9, 18-22, and C-terminal amino acids of the A β40

respectively; 5 ml of CSF was used for each sample. Antibodies were cross-linked to

250 µl magnetic Dynabeads (sheep anti-mouse, IgG) and added to the CSF, followed by

50 µl 2.5 % Tween-20 (Bio-Rad, Hercules, Calif., USA). After washing and purification

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using the KingFisher magnetic particle processor, Aβ peptides were eluted using

0.5 % FA. The collected eluents were speedvac-dried, freezed, shipped in dry ice to

Boston University, and kept at -80 oC until use.

5.2.3 On-line nanoLC - ETD MS

Dried peptides were dissolved in a solvent containing various amounts of ACN

(1 - 50 %), H 2O (99 - 50 %), and FA (0.1 - 8 %), vortexed for 2 - 60 min, and injected

with or without further dilution.

Direct infusion MS along with on-line nanoLC – ETD – MS analyses were

performed on an LTQ-Orbitrap XL spectrometer with ETD capability (Thermo Scientific,

San Jose, CA, USA) coupled to a Waters NanoAquity (Milford, MA) and a TriVersa

NanoMate system (Advion Biosciences, Ithaca, NY) for nanoESI. For online LC-MS

analysis, samples were first passed through a C18 trapping column for clean up and

desalting (Symmetry C18: 180 µm x 20mm, with 5 µm particle size), followed by

separation on an analytical reversed-phase column (BEH130 C18: 150 µm x 100mm,

with 1.7 µm particle size, Waters Corporation, Milford, MA). Mobile phases were

1 % ACN, 99 % H2O, 0.1 % FA (A), and 99 % ACN, 1 % H2O, 0.1 % FA (B). The

separation was performed using various gradients starting at 5 - 45 % phase B and

increasing up to 60 - 80 % phase B in 45, 52, and 90 min at 0.5 µl/min flow rate. Peptide

standards were injected in solutions at 5 – 45 % B composition in accordance with the

starting conditions of the separation in the amount of 200 fMol – 1 pMol, at 0.5 - 5µl injection volumes. For direct infusion nanoESI analysis, peptides were dissolved in

50:50:0.1 ACN:H 2O:FA.

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A typical MS/MS scan cycle consisted of one full FTMS scan (300-2000 m/z )

acquired at a resolution of 60,000 at m/z 400 followed by MS/MS of the three most

abundant ions in the full MS scan acquired at a resolution of 30,000 with the low mass

limit set to 100 m/z . MS/MS was performed using ETD with supplemental activation enabled (En = 15). ETD reagent (fluoranthene) accumulation time was set to 250 ms, and ETD reaction time to 90 ms. The MS/MS isolation width was set to 5.0 m/z and the normalized collision energy to 30. Singly charged ions were excluded. The results were analyzed by the Xcalibur 2.0.7 software.

5.3 Results and Discussion

5.3.1 NanoESI of A β standards

In order to successfully develop a method for analysis of biological samples, it is always a good idea to start with the standards due to the limited amount of sample available to the researcher. Synthetic amyloid peptides are readily available because of the great interest in AD, and the extensive endeavour to understand the disease and to find its cure. A β peptides are known to be very hard to work with due to its amyloidogenic properties: high hydrophobicity along with the “stickiness” of the longest peptides, such as A β40 and A β42, makes method development challenging.

Nonetheless, it was shown in the past that A β peptides could be analyzed by ECD, ETD, and related techniques, and the isoAsp can be differentiated. 202, 216, 217

At first, A β40 standard peptide was nanosprayed on the LTQ-orbitrap in direct

infusion mode (Figure 5.1) to check the sample integrity. The signal of A β40 sprayed at

500 fM concentration was very strong (the absolute intensity was ~10 7), and molecular

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ions in 4+, 5+, and 6+ charge states were observed. In addition, the spectrum contained

a small fraction of doubly charged A β26-40 peptide, which most probably originated from

the impurity in synthetic peptide standards. The sequence of A β26-40 was confirmed by

ETD. The ETD parameters were adjusted for the method and a mock LC-MS method

was run during direct infusion of the standard to further test it.

Figure 5.1 NanoESI mass spectrum of the A β40 standard peptide. Note the presence

of the A β26-40.

5.3.2 On-line nanoLC - ETD MS analysis of the standards

After passing through the column, A β40 standard was no longer detected. This could possibly result from the poor solubility of this large A β peptide in solvent with low organic content, which prevented it from entering the column. To address this issue, various starting conditions of separation were applied, with increasing ACN and FA contents. After multiple attempts of changing experimental parameters, such as solvent, gradient, trapping conditions, etc., A β40 was still not detected in the LC-MS mode. It was found, however, that injecting at ≤ 20 % ACN may be problematic for sample 109

introduction, as the peptide standard prepared in solvents containing 20 % ACN also gave very weak A β40 signal in the direct infusion mode. Increasing the ACN concentration of the same sample by simply adding ACN to the autosampling vial improved the signal regardless of the sample dilution. Nonetheless, even injecting A β40 onto the column in 50 % ACN did not improve its detection in the LC-MS mode.

It must be noted that the smaller peptide A β26-40, which was present in the direct infusion mode, was also detected in LC-MS runs (Figure 5.2). The elution peak width of A β26-40 was fairly broad, which may be due to its strong interaction with the surface of the C18 column. Indeed, this peptide contains the most hydrophobic part of the A β40 as shown on the hydrophobicity chart of the A β40 (Figure 5.3). Similarly,

Aβ40 interaction with the stationary phase may be so strong that it could precipitate on the surface without ever being released. It is also possible that A β40 peptide degrades on the column into smaller peptides, such as A β26-40; however, this is very unlikely and, to the best of our knowledge, has not been reported in the literature.

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2+ [Ab 26-40 ]

m/z Figure 5.2 LC-MS run of A β40, 200 fMol injected. On top is the total ion chromatogram

(TIC). In the middle is the base peak chromatogram for the 707.90-707.92 m/z region, corresponding to the doubly charged ions of A β26-40 peptide as confirmed by ETD, which is shown on the bottom.

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Residue Figure 5.3 Hydrophobicity index of the Aβ40 peptide, generated by GPMAW 5.02.

5.4 Future concepts

LC-MS analysis of the CSF samples has been postponed until successful analysis of the Aβ standards is demonstrated. It is well known that Aβ peptides are difficult to work with; however, they have been successfully analyzed by LC-MS before by various groups. The poor Aβ peptide recovery may be caused by the slight difference in the system used in the current experiment, which needs to be addressed before proceeding further. In particular, switching to a less hydrophobic column that uses shorter chain stationary phase packing material, such as C8, or C4, would be suggested, although this may worsen the separation of peptide mixtures. Further, starting with slightly shorter Aβ peptides (less hydrophobic) for testing might help.

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Chapter 6

β-Peptide analysis by ExD tandem MS

6.1 Introduction

Isoaspartic acid (isoAsp) is a β-type amino acid that has one extra CH 2 group in

the polypeptide backbone, and one fewer on the side chain compared to Asp. As

discussed previously electron activation dissociation (ExD) of isoAsp containing peptides

generates signature fragment ions ( c+ 57 and z•-57) at the positions of the isomerized

residues, allowing differentiation from the non-modified residues. These ions are formed

by the Cα-Cβ backbone bond cleavage. In peptides consisting solely of α-amino acid residues, there is no such bond within the backbone. Fragments produced upon Cα-Cβ

bond rupture are unique to β-amino acid residues and could be used to locate the

position of the β-amino acid if observed. The result from the isoAsp experiment

suggested a possible extension of the method to characterize peptides containing other

β-type amino acids. 216, 218, 219

Circular dichroism spectroscopy, X-ray crystal-structure analysis and nuclear magnetic resonance spectroscopy are primarily used to study the structure of

β-peptides. 138, 140, 142, 144 Radioactive labeling, high performance liquid chromatography

(HPLC) and matrix assisted laser desorption and ionization (MALDI) mass spectrometry

(MS) have also been applied to monitor β-peptides in tissues. 141, 143 However, a fast and

accurate MS based method capable of distinguishing α from β and β2 and β3 type amino

______1This chapter has been partially reproduced with permission from Sargaeva, N. P.; Lin, C.; O'Connor, P. B., Unusual fragmentation of beta-linked peptides by ExD tandem mass spectrometry, Journal of the American Society for Mass Spectrometry , 2011 , 22 , 480-491. 113

acids would be of a great utility for characterization of β-peptides. In this chapter the

potential of the ExD based tandem MS methods to differentiate β-amino acid containing

peptides from their alpha analogues, as well as β2 from β3-type amino acids are

addressed. In general, the results are in good agreement with the findings of recent

electron capture dissociation (ECD) and electron transfer dissociation (ETD) studies of

small β-peptides 220 and ε-peptides. 221 In the present study, in addition to simple model peptides such as Q 06 and substance P, a more complicated system of Puma BH 3

peptide analogues of 26 amino residues is investigated. Furthermore, charge state

dependence of the fragment appearance is considered. An alternative mechanism of

ion-electron reaction induced dissociation of peptides within β-amino acid residues is

discussed.

6.2 Experimental section

6.2.1 Peptides and reagents

The Q 06 β-peptide ( β2Vβ2Aβ2Lβ3Vβ3Aβ3L) was kindly provided by Prof. D.

Seebach, and Dr J. Gardiner, ETH Zurich, Switzerland. C-terminally amidated

Substance P with two amino acids modified to β3-type amino acids (RPKP βQQFFG βLM)

was custom synthesized by AnaSpec (San Jose, CA, USA). Puma BH 3 pro-apoptotic

protein analogue I ( βEEQ βWARE βIGA βQLRR βMAD βDLNA βQYE βRR) and analogue II

(βEEQW βARE βIGAQ βLRR βMADD βLNA βQYER βR) were kindly provided by the group

of Prof. S. Gellman at the University of Wisconsin (Madison, WI, USA), where β

indicates β3-type amino acids. Other reagents: non-modified substance P, (2-

Aminoethyl)-trimethylammonium chloride hydrochloride (cholamine), triethylamine

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(TEA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis,

MO, USA); 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate

(HBTU) from Novabiochem (La Jolla, CA, USA); and 1-Hydroxybenzotriazole (HOBt)

from AK Scientific, Inc. (Palo Alto, CA, USA).

6.2.2 Sample preparation

Cholamine reaction. Covalent attachment of cholamine to the Q 06 β-peptide was

222 performed as previously described. Briefly, 0.1 µmol of Q 06 β-peptide was treated sequentially with 10 µl of 200 mM HOBt in DMSO, 200 µl of 100 mM cholamine in

DMSO containing 200 mM TEA, and 10 µl of 200 mM HBTU in DMSO. The sample was left to react overnight at room temperature, and purified using a ZipTip C 18 solid phase micropipette extraction column before MS analysis.

6.2.3 Mass Spectrometry

Most ECD experiments were performed on a custom built triple quadrupole

Fourier transform ion cyclotron resonance mass spectrometer (qQq-FTICR MS) with a nanospray source and a 7 T actively shielded magnet. 100, 102 Samples were nanoelectrosprayed (nanoESI) (50 nl/min, room temperature) at 5 µM concentration in

50:50 MeOH:H 2O with 1 % formic acid. Ions were isolated in the first quadrupole Q 1,

accumulated in the second quadrupole Q 2, and transmitted into the ICR cell where they

were irradiated with electrons emitted from an indirectly heated dispenser cathode

(Heatwave, Watsonville, CA, USA) for ion fragmentation. The following ECD and

electron ionization dissociation (EID) parameters were employed: electron irradiation

time 35-100 ms, cathode potential -0.2 - 1.2 V (ECD), - 18 V (EID). Acquired spectra

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were zerofilled twice, internally calibrated, and analyzed manually using BUDA (Boston

Daltonics, Billerica, MA, USA) with 12 T actively shielded magnet. Electrospray was applied for enhanced production of triply charged ions.

ETD spectra with supplemental activation were acquired on an amaZon Ion Trap instrument (Bruker Daltonics, Billerica, MA, USA) using fluoranthene as the ETD reagent. Peptides were electrosprayed (2 µl/min, glass capillary temperature 220 ◦C) at

1 µM concentration in 50:50 MeOH:H 2O with 1 % formic acid using an Apollo II ion source. Data acquired on solariX and amaZon were analyzed using Bruker’s ESI

Compass DataAnalysis 4.0 software.

6.3 Results and Discussion

6.3.1 Q 06 beta peptide β2Vβ2Aβ2Lβ3Vβ3Aβ3L.

Although the Q 06 β-peptide only contains six β-amino acid residues, it can form helical secondary structures. 140, 144 This small and relatively simple peptide contains both β2 and β3 type amino acids, making it potentially an ideal system to study for the

+ differentiation of β2 and β3 amino acids. However, only singly charged ions [M+H] were detected in all ESI/ECD experiments, either with the nanospray or with the electrospray ionization sources (Figure 6.1.a), which is likely due to the lack of basic amino acid residues within the peptide sequence. Since ECD and ETD are accompanied by charge neutralization upon the electron capture or transfer, they cannot be performed on singly charged ions as the products would be neutral and undetectable. In this case, other

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fragmentation techniques could be applied that do produce fragments from singly charged ions such as collisionally activated dissociation (CAD) 50 or infrared multiphoton

35 dissociation (IRMPD). Expectedly, IRMPD of the Q 06 beta peptide resulted in b and y fragments with no information on the position of the modifications (Figure 6.1.b). 218

Additionally, Electron Ionization/Impact Dissociation (EID) can generate ECD type

78 fragments from singly charged molecular ions, as well as Cα-Cβ cleavage for isoaspartic acid. 202 However, only b and y fragments were detected in EID mass spectra

216 • of the Q 06 β-peptide (Figure 6.1.c). The lack of c and z fragments could be due to the low fragmentation efficiency of EID in this particular experiment. Thus, the well established ECD method would seem to be a better approach, but it requires an increase in the number of charges on the peptide.

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m/z

Figure 6.1 Q06 beta peptide β2Vβ2Aβ2Lβ3Vβ3Aβ3L: a) ESI, b) IRMPD, c) EID. Asterisk

indicates noise peaks.

6.3.2 Charge increase

Three different approaches were applied in order to enhance the formation of higher charged molecular ions. These include the addition of nitrobenzyl alcohol or calcium salt into the ESI solution, and the covalent attachment of the cholamine tag to the peptide.218, 222-225 Although doubly charged ions were observed in all three cases,

only the cholamine reaction produced sufficient ions for ECD analysis. Inefficiency of

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other methods could be due to the lack of a preferred calcium ion binding site and

potentially low charge stabilization in the peptide. Cholamine reacts with the carboxylic

acid and was attached to the peptide at its C-terminus. The second charge could be

provided by the protonation of the N-terminal amine. Doubly charged species

[M•Ch++H]2+ were readily observed in the ESI MS, isolated, and further subjected to ECD

(Figure 6.2).

Figure 6.2 ECD of Q06•cholamine.

Similar experiments of C-terminal charge tag attachment to α-peptides were done by Hunt’s group.226 Their result demonstrated increased formation of z ions. In the

current study, no z fragments and only one ECD-type fragment, c5, was observed for the

Q06 β-peptide. The electron capture at the quaternary ammonium provides an abundant loss of the neutral trimethylamine 59.0735 = N(CH3)3, leaving the radical on the C-

terminal methylene group (Figure 6.2). This is expected to further induce radical initiated

reactions and cleavages, such as the loss of ethylene. Although charge neutralization

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on the quaternary ammonium may not lead to backbone cleavages, 227 electron capture at the protonated N-terminal amine of the doubly charged Q 06 peptide should result in

backbone fragmentations. Further, the recombination energy of the protonated N-

terminal amino group is higher than that of the quaternary ammonium as calculated for

+ the singly charged model cations by Jensen et al. (4.3 eV (MeNH 3 ) vs. 3.1 eV

+ 228 (NMe 4 )), making it the preferred electron capture site. However, ECD type fragments

were lacking in the ECD spectrum of the tagged Q 06 peptide, which was dominated by b5

and y fragments which could be merely the result of the residual vibrational excitation. 57

Alternatively, the formation of y fragments could be facilitated by the backbone nitrogen protonation or a rearrangement that transfers a hydrogen atom to the backbone nitrogen. 61, 84, 221 Upon charge neutralization at the tag site and the subsequent tag loss, the excessive vibrational energy could induce the formation of the b5 ion, similar to that

suggested by Cooper et al. in their study of b-ion formation in ECD. 60 Nevertheless, it is

hard to make definitive conclusions why this unusual fragmentation pattern was

observed as two variables were introduced at the same time in the studied system: a

cholamine tag and the incorporation of β-amino acids.

6.3.3 ETD of Q 06 beta peptide

Electron transfer dissociation was further used to analyze the Q 06 beta peptide.

As opposed to the first ESI experiment, a small peak corresponding to the doubly

2+ charged molecular ions, [M Q06 +2H] , was observed, which could be due to the

difference in the ionization sources between two instruments. In particular, the glass

capillary in the electrospray source used in the ETD study was heated to 220 ◦C which

120

could possibly increase the desolvation efficiency and production of higher charged ions

compared to the unheated source used in the previous ECD experiment.

Figure 6.3 ETD of Q 06 β2/β3-peptide.

ETD of the Q 06 beta peptide produced an unusual fragmentation pattern with abundant y and a • fragments, and only two c fragments in low abundance (Figure 6.3).

• The assignment of a 5 ion was ambiguous due to the interference from x4 ion

• [x4 (m/z =497.33), and a 5 (m/z =497.39)], which could not be resolved using the ion trap.

The assignment is probably correct, because x ions are not commonly observed in ECD and ETD, and they were not detected for this peptide in ECD experiments. It is not clear how such abundant y fragments could be formed without a C-terminal charge carrier. It might be due to the backbone nitrogen protonation, similar to the cholamine attached

Q06 β-peptide ECD results. Likewise, the alternative dissociation pathway seems to be

enhanced. Meanwhile, low vibrational energy applied to the charge reduced species to

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increase dissociation efficiency upon electron transfer (smart decomposition) may also

be a reason for enhanced y fragment formation. However, this cannot explain the formation of a• fragment ions as those are radicals species and more likely to be a product of ion-electron reaction. In general, the observed fragmentation pattern for this peptide is not following the usual ETD behavior. ExD of this very simple β-peptide demonstrates a big difference for fragment formation in β-peptides compared to their α-

analogues.

6.3.4 Modified Substance P

In order to better understand the unusual ExD behavior of β-peptides, ECD

experiments were carried out on a well studied system, the substance P peptide, and its

variant which was modified at two positions: glutamine 5 and leucine 10 to β3-homo amino acids. Q 5 and L 10 were specifically selected for modification as they normally

provide abundant c4 and c9 fragments (Figure 6.4.a.), and the expected fragments resulted from the C α-Cβ bond cleavage (discussed below) would not interfere with other

peaks in the spectrum. The introduction of the two extra methylene groups in the

backbone increased the molecular mass of the peptide by 28 Da. Fragment ion mass

shift at 14 Da per additional methylene group was also observed correspondingly. As

was discussed in the introduction, the extra methylene group present in isoAsp leads to

a C α-Cβ backbone bond cleavage and the formation of signature diagnostic fragments.

Similar cleavages were expected for the current modifications which would result in

formation of c4+85 and c9+70 fragment ions. However, not only were these ions absent,

the c4 and c9 fragments also disappeared from the ECD spectrum entirely (Figure 6.4).

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Figure 6.4 ECD of Substance P: a) unmodified, b) modified at Q5 and L 10 to β3-type amino acids.

One may speculate that introduction of the CH 2 group could lead to such a conformational change that would hinder the formation of c4 and c9 fragments; or they may still be formed, but not separated due to the new, possibly tighter hydrogen bonding, although the incorporation of only one extra methylene group is unlikely to create such a big difference in intramolecular hydrogen bonds. As proposed

216, 219 previously, it is more likely that the absence of C α-Cβ bond cleavages resulted from the lack of radical stabilization effect by a sidechain carbonyl group as in isoAsp containing peptides. Further, in α-amino acid residues, the α-carbon radical formed by

N-Cβ bond cleavage can be resonantly stabilized by the neighboring carbonyl (Figure

6.5); in β-amino acid residues, the carbonyl is located further away providing no such

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stabilization, making the N-Cβ bond cleavage not energetically favorable. Within the isoAsp, however, a carboxylic acid on a side chain is located at the close proximity to the backbone β-carbon atom, which can play a stabilization role for the β-carbon radical and thus ensure both the C α-Cβ and N-Cβ bond cleavages. In agreement with this

explanation, in a previous ECD study of γ-glutamic acid containing peptides, N-Cγ, C α-

229 Cβ, and C β-Cγ bond cleavages were all observed. Similar stabilizing effect can be provided by the aromatic structure as well, such as that observed in the β-phenylalanine containing peptides, which was recently reported by Hamidane et al. 57 supporting the

radical stability hypothesis.

Figure 6.5 ECD mechanism within α- vs. β-amino acid residues.

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6.3.5 Puma BH 3 protein analogues

Further ECD analysis was performed on a set of bigger peptides, originally

230 designed to mimic foldamer ligands for the BH 3 recognition cleft of the protein Bcl-xL.

The primary sequence of the two 26-residue α/β-peptide analogues corresponds to a

Puma BH 3 domain (EEQWAREIGAQLRRMADDLNAQYERR). Both peptide analogues

nd rd have amino acid residues modified to β3-homo amino acids after each 2 or 3 residue,

but not all at the same positions. Such a backbone repeat ααβαααβ allows formation of

an α-helix like conformation that helps mimic the original binding behavior of an α-helical

domain. For the purpose of the current study, the two peptide analogues provide an

excellent system for the direct comparison of fragmentation within α- and β-type amino

acids. The ECD spectrum of the 3.3 kDa Puma BH 3 protein analogue I

(βEEQ βWARE βIGA βQLRR βMAD βDLNA βQYE βRR) is shown in Figure 6.6; the ECD

spectrum of the analogue II ( βEEQW βARE βIGAQ βLRR βMADD βLNA βQYER βR) is

shown in Figure 6.7. The spectra show nearly complete sequence coverage with

various types of fragments observed, including a•, y, c, and z• fragments. In agreement

with the modified substance P result, no N-Cβ bond cleavages were observed for the β-

type amino acid residues, but the corresponding α-amino acid residues all provided such

• cleavages. For example, c, and z fragments are observed at the α-Ala 5, α-Leu 12,20 , and

α-Arg 26 positions (analogue I) but not in the β-Ala 5, β-Leu 12,20 , and β-Arg 26 positions

(analogue II) (Figure 6.8, relevant residues are highlighted). The only N-Cβ bond rupture

2+ 2+ was detected for β-Asp provided by the z9 and c17 fragments (Figure 6.6).

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Figure 6.6 ECD of Puma BH3 protein analogue I.

Interestingly, this is a β-homo aspartic acid; i.e. the sidechain carboxylic acid is separated from the backbone β-carbon by one methylene group. Hence, β-Asp side chain carbonyl cannot provide the same stabilization for the Cβ radical as in the native

2+ 2+ isoAsp, and the z9 and c17 fragments were only present at very low abundance.

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Figure 6.7 ECD of Puma BH3 protein analogue II.

Figure 6.8 ECD cleavage scheme of Puma BH3 protein analogues I and II.

As was noted earlier, the Puma BH3 peptides studied here have α-helix like

conformations. It is possible that the N-Cβ bonds can be cleaved but protected from

dissociation due to strong hydrogen bonding within the α-helix as was suggested

previously231 and consistent with the results from ECD mechanistic studies.69, 71 Yet, there were many c and z• fragments present except for those missing within β-residues

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suggesting that the peptide was relatively unfolded, and thus, the hydrogen bonds

should not interfere with the fragment separation. Furthermore, dissociation of the C α-Cβ

bond now seems to be an exception rather than the rule for β-amino acid residues as

only one peak representing the Cα-Cβ cleavage of isoleucine was identified provided by

3+• the z19 -C5H10 fragment ion (Figure 6.6). Indeed, according to Turecek and coworkers 232 who studied the β-alanine N-methyl amide model system, dissociation of

the C α-Cβ bond was slow and in competition with other dissociations from the most

stable composition with a radical located on the C-terminal amide carbonyl. To

conclude, the N-Cβ and Cα-Cβ backbone cleavages are still possible, but do not

represent the dominant channel of fragmentation within β-amino acid residues.

6.3.6 Proposed mechanism

The incorporation of an extra methylene group within the polypeptide backbone

increases the flexibility of the molecule. The conformational change is probably not that

dramatic, but an increase in internal rotations is expected, resulting in decreased steric

hindrance. Thus, the backbone nitrogen may become more exposed for hydrogen

bonding. In addition, elongation of the backbone within the β-residue moves the amide

nitrogen and the following amide carbonyl apart removing the captodative stabilization

effect at the C β. Therefore, the N-Cβ bond dissociation becomes a less favorable process, thus shifting dissociation to other fragmentation channels. One such channel may proceed via backbone nitrogen protonation, leading to increased a• and y fragment

233 ion formation (Figure 6.9) as was proposed in an early ECD paper. The N-Cβ bond

rupture may still occur, creating c fragment and unstable intermediate z• fragment that, if

formed, will probably undergo further rapid dissociation, and is thus not observed.

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However, it is more likely, that upon electron capture the radical at the backbone amide

hydrogen will induce the homolytic cleavage of the peptide bond and further loss of a CO

molecule to produce the more stable a• and y fragment ions. It is interesting to note that,

• unlike the doubly charged Q 06 peptide, the a /y fragmentation channel was not observed in the ECD spectrum of the doubly charged β-substance P variant.

Figure 6.9 ECD mechanism for β-amino acid residues.

A closer look at the Q 06 β-peptide and the modified substance P revealed that,

besides the nature of the amino acids, the clear difference between the peptides is the

presence of basic amino acid residues, which could dictate the sites of protonation and

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thus the sites of electron capture dissociation. The two charges carried by the

substance P peptide would preferably reside at the Arg and Lys sidechains, and are

solvated by the carbonyl groups of the peptide. According to the Cornell ECD

mechanism, 57 upon electron capture at the protonated site, hydrogen migration to

various carbonyl groups would result in N-Cα bond cleavages. Due to arginine being a

poor hydrogen donor, backbone fragmentations are most likely initiated by electron

capture at the protonated N-terminal or lysine sidechain amino group. 65, 73, 234 In the

case of the β-substance P, this process will result in the formation of unstable C β

radicals within the βQ and βL residues, which correlates with their disappearance. On the other hand, in the Q 06 β-peptide, there are no basic amino acids. As was discussed

earlier, the first protonation site would be the N-terminal amine; and the second proton

would be mobile within the polypeptide backbone. Thus electron capture would occur at

the N-Terminus or on the backbone rather than on the side chain and the electron

induced fragmentation of the Q 06 peptide would occur via a different mechanism to that of substance P. Introducing the third charge to the substance P peptide should lead to the protonation of an additional site within the molecule, which is likely one of the backbone amide nitrogen or carbonyls (see below), since N-terminal amine protonation seems unlikely because of the strong columbic repulsions by the nearby protonated Lys and Arg residues. To test this hypothesis, triply charged β-substance P was subject to the ECD analysis (Figure 6.10). Indeed, many a• and y fragments now appeared in the

spectra. Interestingly, they often were slightly higher in abundance in the modified

substance P variant as shown in the inset (Figure 6.10). Nonetheless, all of the a• and y

• fragments were observed in both peptides, except for y2 and a9 , which were exclusively

present in the modified substance P variant. The last two fragments are formed due to

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the cleavages within the β-Leu10 residue. Note that the N-Cβ bond cleavage was not

• observed within this residue as c9 and z2 fragments were still absent (yet present in the non-modified variant). This is consistent with the previous results and supports the proposed hypothesis for the fragmentation within the β-amino acid residues.

Figure 6.10 ECD of triply charged substance P [M+3H]3+: a) unmodified, b) modified at

Q5 and L10 to β3-type amino acids.

The role of backbone nitrogen protonation in ECD has been previously discussed in the literature. In the original ECD study, the a• and y ions were proposed to be formed

from the backbone nitrogen protonated species.233 Backbone nitrogen protonation was also suggested to play a role in the formation of b ions in ECD. Theoretical investigation by the Uggerud group showed that electron capture by nitrogen protonated N-methyl-

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acetamide resulted in rapid amide bond dissociation and production of CH 3CO and

• 235 • NH 2CH 2, corresponding to the b and y ions in peptides. Concomitantly, b ions can

further lose CO to form a• ions. In addition, b ions were present in ECD of peptides

without basic amino acid residues. 62 It was suggested that upon backbone nitrogen

protonation, the peptide bond could be cleaved to form a b•/y ion pair, and the

subsequent intra-complex hydrogen atom transfer within this long-lived ion pair could

lead to the formation of b and y • ions. Backbone amide nitrogen protonation was also

invoked to explain the a•, b , and b• ion formation in nitrated peptides that are either

acetylated at the N-terminus, or lacking basic amino acid residues. 236 In the current

study, however, neither b nor y ions observed were radicals. Thus, these b ions were

most likely formed via the energetic fragmentation of vibrationally excited even-electron

charged reduced species. 60

It should be noted that protonation on the backbone nitrogen is not thermodynamically favorable. 237-239 For instance, for the model system of

N-methylacetamide, the proton affinity of the carbonyl oxygen was calculated to be

~60 kJ/mol higher than that of the backbone nitrogen. 238 This is in agreement with the

study of the dipeptide Lys-Gly, where the carbonyl oxygen protonated species was

calculated to be ~45 kJ/mol more stable than the backbone nitrogen protonated

species. 239 Based on these results, peptide fragmentation via protonation of the

backbone nitrogen would seem unlikely. However, theoretical investigations have

usually been done on very small model systems, which often do not possess extensive

intramolecular interactions such as hydrogen bonding and salt bridges that are expected

to play a more important role in the fragmentation of larger peptides. Charge solvation

by nearby backbone and sidechain groups could appreciably change the relative

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stabilities of different protonated species. In β-peptides, the elongation of the backbone

may make it better positioned (due to less steric hindrance by the adjacent side chain)

for hydrogen bond formation. The addition of an extra methylene group in the backbone

can also slightly increases the gas-phase basicity of the amide nitrogen, as the electron

withdrawing carbonyl group is replaced by the electron donating alkyl group. Further,

electron capture by the precursor ion could increase its internal energy considerably (by

several eVs), and less favored protonation sites may become significantly populated as

the system relaxes from the initial Rydberg state to low lying electronic states. In other

words, the proton may initially reside on the carbonyl oxygen, but could migrate to the

backbone amide nitrogen upon excitation. This argument is similar to the one used in

the mobile proton model to explain the low energy CAD fragmentation behavior of

peptide ions, 237, 240 where it was proposed that the proton initially resides on the

thermodynamically more stable sites, such as the lysine sidechain or backbone oxygen,

and later migrates to the less favorable sites, including the backbone amide nitrogen

upon collisional activation to facilitate fragmentations. Finally, for the β-linked peptides

studied here, electron capture at the protonated oxygen site cannot lead to “normal” c/z

fragmentation due to the radical instability, which may further drive the migration of the

proton to the less preferable backbone nitrogen site that could lead to the formation of a•

and y ions upon electron capture. In general, the abundance of these unusual ECD

fragments was fairly low, as expected from the low population of nitrogen protonated

species (Figure 6.10), but they were nonetheless present in competitive abundance

when the primary ECD fragmentation channel was blocked within β-amino acid residues.

Further experiments are needed to test the proposed mechanism. In addition, basicity measurements and theoretical investigations specifically for β-amino acid

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containing peptides could provide a better understanding on how these a• and y type

fragments are formed.

6.4 Conclusion

Various peptides containing β-amino acid residues were analyzed in this study.

Remarkably, N-Cβ bond cleavages were rare within the β-residues, and C α-Cβ cleavages

were seldom observed providing no evidence of the β-residues in spite of previous

results of the isoaspartic acid. Furthermore, no distinct difference was found for the

• fragmentation within β2 vs. β3-type amino acid residues. Meanwhile, a and y fragments

were often produced at β-residues, particularly for the bigger peptides with α-helical like

structures. The lack of z• and c fragments and increased a• and y fragment formation could imply the presence of β-residues in the peptide; however, this is a poor signature,

because of the normal appearance of a• and y fragments in ExD spectra of α-peptides,

and various other reasons that can contribute to the disappearance of z• and c

fragments. Thus, currently, ExD methods cannot be used to reliably differentiate α- from

β- or β2 from β3 type amino acids.

The introduction of one extra methylene group into the polypeptide chain destabilizes the C β radical formed by the N-Cβ bond rupture making this channel of

fragmentation less favorable. Thus the fragmentation occurs via alternative channels.

The dominant products appear to be the a• and y fragments, with the exception when the

side chain of the β-residue can provide radical stabilization for the formation of the z• and

c fragments. It is suggested that appearance of such fragments may require protonation

on the backbone amide nitrogen, which is further supported by the charge state-

dependent study of modified substance P peptide. The fragmentation mechanism for β-

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peptides has been proposed via backbone nitrogen protonation similarly to what was

originally proposed for the ECD of α-peptides.

The minor ECD pathway of a• and y fragment formations was little studied.

Future studies of this fragmentation pathway would help with our understanding of the

fragmentation of the β-amino acid containing peptides. Further ExD studies, as well as

computational studies and kinetic analysis specifically for the β-amino acids are needed for better characterization of β-peptides.

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Chapter 7

In-Source Decay for β-Peptide Analysis

7.1 Introduction

In-source decay (ISD) during the matrix assisted laser desorption ionization

(MALDI) process can produce abundant ECD-like fragmentation resulting from the

88 cleavage of the N-Cα bond on the polypeptide backbone. The ISD process is believed

to be mediated by hydrogen radical transfer from the matrix to the analyte ions

generating aminoketyl radicals to produce c- and z- type fragments (Figure 7.1.a). The

radical nature of the ISD method and its similarity to the ECD process suggest that

characterization of β-peptides may lead to no or little fragmentation of C α-Cβ bond due to the lack of C β radical stabilization based on the results described in the previous chapter.

It was recently reported that some novel matrices, such as 5-formylsalicylic acid (5-FSA) and 5-nitrosalicylic acid (5-NSA), can accept a hydrogen from the analyte ions. 91, 92 In

this case, the hydrogen abstraction from the peptide results in the formation of an

oxidized peptide containing a radical site on the backbone amide nitrogen with

subsequent radical-induced cleavage at the C α-C bond, leading to the formation of a-

and x-type fragments (Figure 7.1.b).

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Figure 7.1 Formation of the ISD fragments: (a) c/z-type via hydrogen attachment and (b)

a/x-type via hydrogen abstraction.

In β-amino acids, because of the presence of an extra methylene group between

the C α and the carbonyl C, it is possible that amide nitrogen radical driven fragmentation

would result in the cleavage of the C α-Cβ bond. Thus, it is hypothesized that the use of hydrogen accepting matrices in ISD may lead to formation of diagnostic fragments for β- amino acids.

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7.2 Experimental section

7.2.1 Peptides and reagents

C-terminally amidated Substance P with two amino acids modified to β3-type amino acids (RPKP βQQFFG βLM) was custom synthesized by AnaSpec (San Jose, CA,

USA). Non-modified Substance P was purchased from Sigma-Aldrich (St. Louis, MO,

USA); Formic Acid (FA) was purchased from Thermo Scientific (Rockford, IL, USA).

HPLC grade Acetonitrile (ACN) and Methanol (MeOH) were obtained from Fisher

Scientific (Fair Lawn, NJ, USA). Deionized water was purified by a Millipore Milli-Q

Gradient system (R=18.2 MΩ cm and TOC = 9 – 12 ppb) (Billerica, MA, USA). 5-

Aminosalicylic acid (5-NSA, or 2-hydroxy-5-nitrobenzoic acid) matrix was obtained from

Sigma Aldrich (St. Louis, MO, USA).

7.2.2 Sample preparation

Peptides were dissolved in 50:50 ACN:H 2O with 0.1 % FA at a concentration of

20 pmol/µl. 5-NSA matrix was dissolved in the same solvent at a concentration of 20 mg/ml. Sample was deposited onto a stainless steel MALDI target in a “sandwich” manner: 1 µl of matrix solution followed by 1 µl of analyte solution and then again 1 µl matrix solution on top, letting the droplet dry each time before spotting the next layer.

7.2.3 Mass Spectrometry

The in-source decay fragmentation was performed on a solariX hybrid Qh-FTICR instrument at the Bruker facility (Bruker Daltonics, Billerica, MA, USA) with a 12 T actively shielded magnet. The laser power was 30 %, 1000 Hz laser frequency, 300

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laser shots per scan, 50 scans were accumulated for each spectrum. Data were analyzed using dataAnalysis 4.0 software.

7.3 Results and Discussion

7.3.1 Diagnostic fragments for the C α-Cβ bond cleavage

Figure 7.2 MALDI-ISD-FTICR mass spectrum of modified substance Pusing 5-NSA matrix. Data was acquired at the Bruker Daltonics facility, Billerica, MA.

MALDI-ISD experiment was performed on a synthetic substance P analogue with two β-homo-amino acids incorporated at the glutamine-5 and leucine-10 positions. The resulting ISD spectrum is presented in Figure 7.2. The peptide structure is illustrated on top, and the insets show the diagnostic ions, a5-CH 2 and a10 -CH 2, for the two β-amino acid residues present. Characteristic for hydrogen accepting matrices, 91 a peak corresponding to the loss of two hydrogen atoms is also indicated in spectrum

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(a10-CH2-2H). The MALDI spectrum of the blank 5-NSA matrix was acquired at the

same conditions as a control (insets).

Figure 7.3 MALDI-ISD-FTICR mass spectrum of the β-Q06 peptide. Insets show the

diagnostic ions for β2-valine-1, and β3-leucine-5 amino acids with corresponding region

of a blank matrix as a control on top. * indicates abundant matrix cluster ions, **

indicates unidentified chemical noise peaks.

Another β-peptide, β-Q06, was analyzed with MALDI-ISD (Figure 7.3). For this small peptide, it was very difficult to obtain doubly charged precursor ions required for

ECD analysis (Chapter 6). Thus, this peptide seems to be a good case to test the ISD performance. Interestingly, similar to the ECD and ETD data, the ISD spectrum of β-Q06

contains mostly b- and y- fragments, which could be the result of post-source decay

mediated by collisions in the hot MALDI plume owing to the high laser power used for

90 the ISD experiment. Nonetheless, as a result of the ISD process, two diagnostic Cα-Cβ

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• cleavages were observed, x5 +C 4H8 and a5-CH 2. β-Q06 contains both β2 and β3 amino

acids, and if one can produce the C α-Cβ bond cleavage, it should be possible to distinguish β2- from β3-type amino acids. Indeed, a “switch” of the side chain from the Cα

• to C β carbon in β3-type amino acids would provide diagnostic fragments x +[side chain]

• and a-[side chain], whereas for β2-type amino acids, such fragments would be x +CH 2

and a-CH 2. One diagnostic ion of each complementary pair was observed for the β2-

valine-1 and β3-leucine-5 residues, respectively, with their complementary ions having too small m/z value to be detected. In addition, similar to the substance P result, double

hydrogen losses were also observed for β-Q06 . Mechanisms for the formation of

diagnostic fragments within β-amino acids in ISD using hydrogen accepting matrices are

proposed in Figure 7.4.

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Figure 7.4 Upon the loss of a hydrogen, amide nitrogen radical would cleave the Cα-Cβ

• bond producing diagnostic a-14 and x +14 in the case of the β3-type amino acid (a) and

• a-[side chain] and x +[side chain] in the case of the β2-type amino acid (b).

7.3.2 Advantages and disadvantages of the proposed method

One of the limitations of the ISD method is that fragmentation occurs during the

MS1 event, and the precursor ion may not be isolated prior to fragmentation as is

possible in most tandem MS methods. Therefore, sample purity is essential for

successful data interpretation and sample complexity must be reduced, ideally to single

species with, for example, HPLC prior to the MALDI-ISD analysis. The matrix amount

necessary for successful desorption, ionization, and ISD fragmentation is high

(~10 mg/µl). Matrix ions, matrix clusters and analyte-matrix adducts are usually

abundant in MALDI spectra and represent dominating peaks below 1000 m/z limiting the 142

analysis of smaller analyte molecules due to the peak interference (Figures 7.2 and 7.3).

Therefore, blank matrix spectrum must be used as a control for comparison. In some instruments, such as FTICR and Q-o-TOF, the longer delay between ion formation and detection allows some matrix clusters to fall apart leading to less matrix interference.

Higher mass resolving power instruments are highly desirable for this kind of experiments for confident assignment of matrix/analyte peaks.

One of the advantages of the ISD method is its ability to generate ECD-type fragments from singly charged precursor ions, which cannot be analyzed by ECD and most ECD-related techniques due to the charge reduction upon electron capture.

Importantly, ISD can produce specific cleavages of the N-Cα, C α-C, and C α-Cβ bonds of the peptide backbone without loss of the side-chains and PTMs, providing a valuable tool for the analysis of biological samples.

7.4 Conclusion

The in-source decay fragmentation was applied toward the analysis of β-linked amino acid containing peptides. The cleavage of the C α-Cβ bond on the polypeptide backbone was observed providing diagnostic fragments for the β-amino acids. In addition, it was possible to differentiate β2-type from β3-type amino acids using ISD, which was not possible with ECD. However, for some C α-Cβ bonds present, diagnostic fragments were not detected. The efficiency of such cleavage may depend on the peptide sequence and various parameters of the experiment, such as the laser power, analyte - matrix concentration ratio, and others. Thus, the method needs to be further optimized for the best results. Nonetheless, ISD seems to be a promising tool for the analysis of β-peptides.

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Chapter 8

Conclusion and Future Perspective

8.1 Conclusion

Deamidation and isomerization are two common post translational modifications

(PTMs), which accumulate with time and have been associated with a number of diseases. These modifications have garnered a lot of attention from researchers in various biospheres. Much progress has been made toward the analysis and characterization of these PTMs in peptides and proteins, although it still remains a challenging task. Electron capture dissociation (ECD) technique is one of the great recent achievements in this area. In this thesis, the ECD method was applied to characterize specific species, and new and improved methods were suggested in general analysis of deamidation and its products.

Amyloid β (A β) peptides are the major components of the vascular and plaque amyloid filaments in individuals with Alzheimer’s disease (AD). Isoaspartic acid (isoAsp) was detected in A β peptides in the brain of AD patients and, thus, is related to the pathology of AD. In this thesis, ECD was applied to analyze amyloid β protein fragments to differentiate isomerized Asp residues. The most aggregative fragment A β42 was successfully characterized by top-down ECD-MS/MS approach. It was further shown that electron ionization/impact dissociation (EID) can also produce diagnostic fragment ion on the example of the tryptic peptide A β17-28. This method would benefit the analysis of singly charged small or non-polar peptides, which is not possible with the

ECD technique, because of the charge neutralization. Further, N-terminal isoAsp was differentiated in A β and other peptides by ECD and electron transfer dissociation (ETD),

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collectively known as electron activated dissociation (ExD) methods. New diagnostic fragment ions for N-terminal isoAsp were proposed and demonstrated in peptides with free N-terminus and acetylated one.

Mixed A β and other peptides containing Asp, isoAsp, and Asn at one or two positions, including the N-terminal modification, were characterized using reversed- phase (RP) high performance liquid chromatography (HPLC). Although it was shown that HPLC separation alone may lead to erroneous assignment of peptide isomers because of the sequence and separation parameter dependent changes in elution orders, it is an important complementary technique in proteomics analysis. Predictive chromatography was suggested as an aid to isoAsp-containing peptide separations in combination with tandem MS and when ExD methods are not available. Further, the cerebrospinal fluid (CSF) studies on isomerized A β peptides were initiated and would bring an interesting addition to the amyloid β research field.

Because isoAsp is a β-type amino acid, the interest appeared in other β-amino acids and β-peptides. β-Amino acids differ from their α-analogues by the presence of an additional methylene group on the polypeptide backbone. This difference leads to the formation of a bigger variety of secondary and tertiary structures in β-peptides, yet the functional side-chain groups of amino acids remain the same. Another important property of β-peptides is their resistance to proteolytic degradation in living organisms.

These features make them good candidates for therapeutic agents by mimicking α- peptide’s and protein’s active sites and allowing prolonged circulation in the body.

Despite substantial progress in β-peptide synthesis and understanding of their structure, a fast and accurate analytical method for analysis of β-peptides is lacking. It was proposed in this thesis that ExD methods could be one such technique, based on the

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isoAsp analysis results. However, ExD produced no diagnostic fragment ions at β-

amino acid residue sites. It appears that formation of isoAsp diagnostic ion is driven by

the stability of the C β radical due to the presence of the isoAsp side-chain’s carboxylic

group. Such radical stabilization is absent in other β-amino acids, except for β-

phenylalanine. Although no diagnostic ions were detected, the fragmentation pattern

within β–amino acid residues was somewhat different from that within α-amino acid residues. The characteristic N-Cα bond cleavage was severely diminished or not

observed producing little or no c and z fragments, yet the alternative ECD pathway, in

which a and y ions are formed was activated. These results lead to a better

understanding of the ECD mechanism as well as of other fragmentation methods

producing ECD-like fragments. One such technique is the in-source decay (ISD). It was

proposed that use of hydrogen-accepting matrices in ISD may lead to Cα-Cβ bond

cleavage in β-amino acids, because the resulting radical ion would be stabilized by the polypeptide backbone carbonyl group irrespective of the amino acid’s side-chain. This hypothesis was tested on a few peptides containing β-amino acid residues which showed promising results. β-Amino acid residue-characteristic cleavages of the C α-Cβ

bond were generated providing diagnostic fragment ion peaks in the ISD spectra. In

addition, characteristic to the use of hydrogen accepting matrices in ISD, the

accompanying peaks of double hydrogen loss were also observed, giving an additional

confirmation of the result.

In conclusion, the author believes that the results of the work in this thesis have

brought a valuable addition to the deamidation and related problems in structural

analysis of peptides and proteins by investigating and improving the existing methods of

analysis as well as by introducing a new means for β-peptide characterization.

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8.2 Future Perspective

Despite the great progress in the development of analytical methods for the characterization of deamidation and isomerization and in biological research of these

PTMs, there are ample possibilities for further method improvement and applications.

One such interesting project involves protein L-isoaspartyl O-methyltransferase (PIMT) enzyme (registry number: EC 2.1.1.77). PIMT is an isoAsp-repairing enzyme, which catalyzes the conversion of isoAsp back to Asp residue. Deamidation as well as isomerization reaction are highly sequence dependent processes. Deamidation rates are the fastest for the Asn-Gly sequence, followed by the Asn-His, Asn-Ser, and Asn-Ala sequences. It is possible that the specificity of the PIMT enzyme is also sequence dependent. This can be tested by a mechanistic study of isoAsp-containing peptides with varied position of modification within the sequence on the PIMT enzyme reaction rates. In order to do so, one would need to measure the disappearance of PIMT enzyme substrates by, for example, measuring the isoAsp diagnostic fragment abundance in the spectra based on calibrated linear curves of the known isoAsp concentrations.

Proteomic scale analyses of isoAsp peptides have already been performed by means of a “bottom-up” LC-ECD-MS/MS approach. 205 This is a great achievement, although such “bottom-up” analysis has its limitations, such as loss of some peptides because of difference in hydrophobicity in LC separation and ion suppression during ionization process in MS. Further, artifactual deamidation can be introduced to the sample during sample handling. For instance, trypsin digestion commonly used in the bottom-up approach is normally performed at pH ~8 under which condition partial deamidation of Asn in rapid-deamidating sequences such as AsnGly and AsnSer could

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occur if performed over 4 hours. On the contrary, in a top-down mode, these limitations

are eliminated because the single protein characterization should yield a 100 %

sequence coverage and does not require prior digestion. However, the analysis of intact

proteins can be quite challenging and has its own limitations such as disrupting the

protein 3D structure inside the mass spectrometer for high efficiency fragmentation.

Some preliminary work has been done toward this direction but further development

would be needed. 241

Preliminary data have been acquired on the analysis of β-peptides by tandem

MS. Although for most β-amino acids studied, diagnostic fragments were not observed using ExD methods, some amino acids yield those in a low abundance. Thus, sequence dependence in formation of diagnostic fragments in β-peptides would provide a better understanding of the fragmentation mechanism. Additional ExD studies, as well as computational studies and kinetic analysis specifically for the β-amino acids are needed for better characterization of β-peptides.

Further, preliminary studies exploring the utility of ISD in β-peptide analysis have been performed, but analysis of a larger number of samples would be necessary for complete method characterization. In biological research, it is important to get quantitative information as the outcome of the analytical investigation. This can be done by using either isotope labeling technique or by creating linear curves of known concentration model peptides similarly as was done for the isoAsp analysis. 178

Other radical-induced fragmentation methods, such as ultraviolet

photodissociation (UVPD), 24 could bring a new complementary method for analysis of

both deamidation/isomerization products and β-amino acid-containing peptides.

148

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(231) Skurski, P.; Sobczyk, M.; Jakowski, J.; Simons, J., Possible mechanisms for protecting N-C-alpha bonds in helical peptides from electron-capture (or transfer) dissociation, International Journal of Mass Spectrometry , 2007 , 265 , 197-212.

(232) Yao, C. X.; Syrstad, E. A.; Turecek, F., Electron transfer to protonated beta- alanine N-methylamide in the gas phase: An experimental and computational study of dissociation energetics and mechanisms, Journal of Physical Chemistry A, 2007 , 111 , 4167-4180.

(233) Zubarev, R. A.; Kruger, N. A.; Fridriksson, E. K.; Lewis, M. A.; Horn, D. M.; Carpenter, B. K.; McLafferty, F. W., Electron capture dissociation of gaseous multiply-charged proteins is favored at disulfide bonds and other sites of high hydrogen atom affinity, Journal of the American Chemical Society , 1999 , 121 , 2857-2862.

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Curriculum vitae

Nadezda P. Sargaeva

Address: 6103 Stearns Hill Road, Waltham, MA 02451

Email: [email protected]

Year of Birth: 1981

Education

Ph.D. Biochemistry Boston University School of Medicine, Boston, MA, anticipated May 2012 Thesis title: “Deamidation and related problems in structural analysis of peptides and proteins” Advisor: Professor Peter B. O’Connor

M.Sc. and B.S. in Physical Electronics St-Petersburg State Polytechnical University, Russia, 2002, 2004 Concentration: Physics of Medical Technologies Advisor: Professor Oleg Y. Tsybin

Additional education: Integrated course of Liquid Separation and Mass Spectrometry, Uppsala University, Sweden, 2003 CUSO PhD Summer School “Frontiers in Mass Spectrometry”, EPFL, Switzerland, 2009

Research and Professional Experience

2006 Research assistant, Chemistry department, Joensuu University, Finland. Prof. Pirjo Vainiotalo 2004-2006 Graduate Research Assistant, St-Petersburg State Polytechnical University, Russia. Prof. Oleg Y. Tsybin 2003-2004 Exchange Graduate Research Assistant, the Angstrom Laboratory, Uppsala University, Sweden. Prof. Per Hakansson 2002-2003 Research Assistant, BioAnalytical Center “CSU Analytical Spectrometry”, St.-Petersburg, Russia

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Selected Publications

• Sargaeva, N.P ., Yu, X., Costello, C. E., O’Connor, P.B., Lin, C., et.al. In source decay fragmentation can cleave the C α-Cβ bond in peptides producing the diagnostic fragment for isoaspartic acid as well as β-amino acid residues (manuscript in preparation). • Sargaeva, N.P ., Lin, C., O’Connor, P.B., Identification of the N-terminal isoaspartic acid residue in peptides, Analytical Chemistry , 2011, 83 (17), pp 6675–6682. • Sargaeva, N.P ., Lin, C., O’Connor, P.B., Unusual Fragmentation of β-Linked Peptides by ExD Tandem Mass Spectrometry, Journal of the American Society for Mass Spectrometry , 2011 , 22 (3), pp 480-491. • Sargaeva, N.P ., Goloborodko, A.A., O’Connor, P.B., Moskovets, E., Gorshkov, M.V., Sequence-specific predictive chromatography to assist mass spectrometric analysis of asparagine deamidation and aspartate isomerization in peptides, Electrophoresis , 2011 , 32 (15), pp 1962–1969. • Sargaeva, N.P ., Lin, C., O’Connor, P.B., Identification of Aspartic and Isoaspartic Acid Residues in Amyloid beta Peptides, Including A beta 1-42, Using Electron-Ion Reactions, Analytical Chemistry , 2009, 81 (23), pp 9778–9786. • Sargaeva N.P. , Tsybin Y.O., Tsybin O.Y., Hakansson P. “Low-energy electron injection system in Fourier transform ion cyclotron resonance mass spectrometry”, Nauchnoe Priborostroenie (Scientific Instrumentation), vol.15 (4 ), 2005 , 51-55 (publication in Russian).

Honors and awards 2010 - Myrna and Carl Franzblau Student Travel Award, invited speaker to the Uppsala Conference on Electron Capture and Transfer Dissociation, Nara, Japan 2004 - The best scientific diploma work within Universities of Russia

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